Constructs targeting afp peptide/mhc complexes and uses thereof

ABSTRACT

The present application provides constructs comprising an antibody moiety that specifically binds to a complex comprising an AFP peptide and an MHC class I protein. Also provided are methods of making and using these constructs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 15/563,912, filed on Oct. 2, 2017, which is a U.S.national phase application under 35 U.S.C. § 371 of InternationalApplication No, PCT/US2016/025755, filed on Apr. 1, 2016, which claimspriority to U.S. Provisional Application No. 62/142,958, filed on Apr.3, 2015, U.S. Provisional Application No. 62/244,653, filed on Oct. 21,2015, and U.S. Provisional Application No. 62/304,915, filed on Mar. 7,2016, the contents of all of which are hereby incorporated by referencein their entireties.

FIELD OF THE INVENTION

This invention pertains to antibody constructs that specifically bindMHC molecules complexed with AFP peptides, and uses thereof includingtreating and diagnosing diseases.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 750042000102SEQLIST.txt,date recorded: Mar. 16, 2021, size: 66 KB).

BACKGROUND OF THE INVENTION

Cell surface proteins constitute only a small fraction of the cellularproteins and these proteins are often not tumor-specific. Because of theinability to easily penetrate cells, marketed therapeutic monoclonalantibodies (mAbs) recognize these cell surface proteins, most of whichare lineage or differentiation antigens (Milenic, E. D., Curr. Pharm.Des. 8:1794-1764, 2002; Grillo-Lopez, A. J., Expert Rev. AnticancerTher. 2(3):323-329, 2002; Jones, K. L. & Buzdat, A. U., Lancet Oncol.10(12):1179-1187, 2009). In contrast, mutated or oncogenictumor-associated proteins are typically nuclear, cytoplasmic orsecreted, which are currently best addressed either by small moleculedrugs, or in the case of secreted proteins, hardly addressed asanti-cancer drug targets (Reddy et al., Expert Opin. Ther. Targets3:313-324, 2012; Takeuchi, K. & Ito, F., Biol. Pharm. Bull.34(12):1774-1780; Roychowdhury, S. & Talpaz, M., Blood Rev. 6:279-290,2011). However, most intracellular proteins can be proteosomallydegraded, processed and presented by MHC molecules on the cell surfaceas T cell peptide epitopes in the context of MHC molecules that arerecognized by T cell receptors (TCRs) (Morris et al., Blood Rev.20:61-69, 2006; Konnig, R., Curr. Opin. Immunol. 14(1):75-83, 2002).Therefore, generating therapeutic mAbs that recognize the secreted orintracellular tumor antigen-derived peptide/MHC complexes on the cellsurface will take advantage of the enhanced specificity and therapeuticpotency offered by mAbs. Recent advances in using phage display togenerate mAbs have made it possible to select agents with exquisitespecificity against defined epitopes from large antibody repertoires. Anumber of such mAbs specific for solid tumor antigens, in the context ofHLA-A01 and HLA-A02, have been successfully selected from phage displaylibraries (Noy et al., Expert Rev. Anticancer Ther. 5(3):523-536, 2005;Chames et al., Proc. Natl. Acad. Sci. USA 97:7969-7974, 2000; Held etal., Eur. J. Immunol. 34:2919-2929, 2004; Lev et al., Cancer Res.62:3184-3194, 2002; Klechevsky et al., Cancer Res. 68(15):6360-6367,2008). More recently, a human mAb specific for human WT1/HLA-A02complex, a well-described T cell epitope, has been shown to inhibitmultiple cancer cell lines and primary cancer cells via Fc-mediatedeffector cell function (Dao et al., Sci. Transl. Med. 5:176ra33, 2013;Veomett et al., Clin. Cancer Res. doi: 10.1158/1078-0432, 2014) incellular assays and in in vivo models.

Alpha-fetoprotein (AFP) is a 69 kD glycoprotein produced in the yolk sacand fetal liver and secreted into circulation. The synthesis of AFPdecreases dramatically after birth and only trace amounts are present inthe adult liver. In normal adult human blood serum, AFP concentration isusually as low as 5-7 ng/ml (Terentiev, A. A. & Moldogazieva, N. T.,Tumour Biol. 34(4):2075-2091, 2013). However, expression of the AFP geneis reactivated in adults during liver regeneration, hepatocarcinogensis,germ cell tumor or in some cases of viral infection (HBV/HCV). Themeasurement of serum AFP, therefore, plays an important role indiagnosis and in monitoring responses to the treatment of AFP-positivecancers. Currently, AFP is considered a “gold standard” amongtumor-specific molecular biomarkers. However, because AFP is not acell-surface protein, targeting AFP has not been a very active area foranti-cancer antibody drug development.

Primary liver cancer is the fifth most common form of cancer worldwideand the second most common cause of cancer-related death. In 2012, therewere about 782,000 new cancer cases globally, and about 746,000 livercancer related deaths(http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx). 90-95% of livercancers are hepatocellular carcinoma (HCC). Chronic liver damage, suchas that caused by chronic hepatitis, liver cirrhosis and fatty liverdisease, is closely associated with the occurrence of HCC. Hepatitisvirus infection (e.g. HBV, HCV), aflatoxin B exposure, alcohol intakeand other metabolic diseases (e.g. obesity and diabetes) are well-knownrisk factors for HCC. The incidence of HCC is high in East Asian andAfrican countries due to the prevalence in HBV and HCV in these regions(Shibata, T. & Aburatani, H., Nat. Rev. Gastroenterol. Hepatol.11(6):340-349, 2014). However, the number of patients infected with HCVhas been rapidly increasing in Western countries, especially in the USAwhere viral hepatitis infection is partly mediated through drug abuse.Meanwhile, the incidences of cirrhosis owing to nonalcoholicsteatohepatitis (NASH) and obesity also increased in Western countries.In the US, HCC is the 9^(th) most common cancer (Vallanueva et al., Nat.Rev. Gastroenterol. Hepatol. 10(1):34-42).

Mean survival of patients with HCC is 3 months from diagnosis. Howeverthis is closely related to the stage of the tumor and the extent ofunderlying liver disease. As only a minority of HCC patients isconsidered suitable for resection and transplantation at the time ofdiagnosis, treatment for the majority of patients with HCC is mainlypalliative. Non-surgical treatments, such as trans-arterialchemoembolization (TACE/TAE), radio-frequency ablation and systemictargeted agent like sorafenib, have been shown to reduce tumor burdenand improve survival rate but these treatments do not eradicate cancercells and the patients frequently relapse. Therefore, the development ofmore effective therapies remains a pressing field of research (Behboudiet al., Liver Int. 30(4):521-526, 2010).

AFP expression is reactivated in approximately 80% of HCC. Immunotherapystudies aimed at generating AFP-specific cytotoxic CD8 T cell responsesthat recognize peptides presented on HCC cancer cell surface by MHCclass I proteins have reported many human AFP peptides as T-cellepitopes (Butterfield et al., J. Immunol. 166:5300-5308, 2001; Pardee,A. D. & Butterfield, L. H., OncoImmunol. 1:48-55, 2012; Butterfield etal., J. Trans. Med. 12:86, 2014; Liu et al., J. Immunol. 177(1):712-721,2006; Mizukoshi et al., Int. J. Cancer 118(5):1194-1204, 2006). Amongthese peptides, FMNKFIYEI (AFP158) is an immunodominant T-cell epitoperestricted by HLA-A*02:01. AFP/HLA-A*02:01 complex inducedpeptide-specific T cells in vitro from normal HLA-A*02:01 donors. TheseAFP158 specific T cells recognized HLA-A*02:01 positive and AFP positivetumor cells in both cytotoxicity assays and IFNγ ELISPOT assays. AFP158was identified by mass spectrometric analysis of surface peptides froman HLA-A*02:01 positive HCC cell line, HepG2, but not from a HLA-A*02:01negative cancer cell line, Hep3B (Butterfield et al., J. Immunol.166:5300-5308, 2001). These data support that AFP158 is indeed processedand presented by HLA-A*02:01 molecules in AFP-positive cancer cells.Therefore, AFP158/HLA-A*02:01 is a good target candidate for mAb cancerdrug development.

Traditional approaches to using AFP as a therapeutic target for treatingcancers that overexpress AFP have relied on using antibodies that targetthe AFP protein or vaccination with various AFP peptides. Antibodiesdirected against the AFP protein have little therapeutic efficacy sinceAFP is not a cell-surface protein, and may be present at highcirculating levels, thus reducing any specific targeting of the antibodyto the cells expressing AFP. While vaccination with variousMHC-restricted AFP peptides and variants thereof has been observed tolead to activation of AFP-specific T cells and increased cytotoxicity ofAFP-presenting cells in vitro, a clinically significant therapeuticbenefit has yet to be observed.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application in one aspect provides constructs (such asisolated constructs) that bind to a complex comprising an AFP peptideand an MHC class I protein (referred to herein as an “AFP/MHC class Icomplex,” or “AMC”). In some embodiments, the constructs (“anti-AMCconstructs”) comprise an antibody moiety (referred to herein as an“anti-AMC antibody moiety”) that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein.

Thus, in some embodiments, there is provided an anti-AMC construct (suchas an isolated anti-AMC construct) comprising an antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein. In some embodiments, the AFP/MHC class I complex ispresent on a cell surface. In some embodiments, the AFP/MHC class Icomplex is present on the surface of a cancer cell.

In some embodiments, the anti-AMC construct comprises an antibody moietythat specifically binds to a complex comprising an AFP peptide and anMHC class I protein, wherein the MHC class I protein is HLA-A. In someembodiments, the MHC class I protein is HLA-A02. In some embodiments,the MHC class I protein is the HLA-A*02:01 subtype of the HLA-A02allele.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theantibody moiety cross-reacts with a complex comprising the AFP peptideand a second MHC class I protein having a different HLA allele than theMHC class I protein. In some embodiments, the antibody moietycross-reacts with a complex comprising an interspecies variant of theAFP peptide and the MHC class I protein. In some embodiments, theantibody moiety cross-reacts with a complex comprising a variant of theAFP peptide comprising one amino acid substitution (such as aconservative amino acid substitution) and the MHC class I protein.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein the AFPpeptide is about 8 to about 12 (such as about any of 8, 9, 10, 11, or12) amino acids in length. In some embodiments, the AFP peptide isderived from human AFP. In some embodiments, the AFP peptide has anamino acid sequence selected from the group consisting of SEQ ID NOs:3-13 and 16. In some embodiments, the AFP peptide has the amino acidsequence FMNKFIYEI (SEQ ID NO: 4).

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theantibody moiety is a full-length antibody, a Fab, a Fab′, a (Fab′)2, anFv, or a single chain Fv (scFv). In some embodiments, the antibodymoiety is fully human, semi-synthetic with human antibody frameworkregions, or humanized.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theantibody moiety binds to the AFP/MHC class I complex with an equilibriumdissociation constant (K_(d)) between about 0.1 pM to about 500 nM (suchas about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10nM, 50 nM, 100 nM, or 500 nM, including any ranges between thesevalues). In some embodiments, the isolated anti-AMC construct binds tothe AFP/MHC class I complex with a K_(d) between about 0.1 pM to about500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges betweenthese values).

In some embodiments, the anti-AMC construct comprises an antibody moietythat specifically binds to a complex comprising an AFP peptide and anMHC class I protein, wherein the antibody moiety comprises: i) a heavychain variable domain comprising a heavy chain complementaritydetermining region (HC-CDR) 1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (such as about any of 1, 2, or 3) amino acidsubstitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (such as about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (such as about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising a lightchain complementarity determining region (LC-CDR) 1 comprising the aminoacid sequence of S/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or avariant thereof comprising up to about 3 (such as about any of 1, 2, or3) amino acid substitutions, and an LC-CDR3 comprising the amino acidsequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereofcomprising up to about 3 (such as about any of 1, 2, or 3) amino acidsubstitutions, wherein X can be any amino acid.

In some embodiments, the anti-AMC construct comprises an antibody moietythat specifically binds to a complex comprising an AFP peptide and anMHC class I protein, wherein the antibody moiety comprises: i) a heavychain variable domain comprising an HC-CDR1 comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 57-66, or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 67-76, or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and anHC-CDR3 comprising (and in some embodiments consisting of) the aminoacid sequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising (and in some embodiments consisting of) the aminoacid sequence of any one of SEQ ID NOs: 90-99, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, an LC-CDR2 comprising (and in some embodimentsconsisting of) the amino acid sequence of any one of SEQ ID NOs:100-109, or a variant thereof comprising up to about 3 (such as aboutany of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 110-119, or a variant thereof comprising up to about5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC construct comprises an antibody moietythat specifically binds to a complex comprising an AFP peptide and anMHC class I protein, wherein the antibody moiety comprises: i) a heavychain variable domain comprising (and in some embodiments consisting of)an HC-CDR1 comprising (and in some embodiments consisting of) the aminoacid sequence of any one of SEQ ID NOs: 57-66, an HC-CDR2 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 67-76, and an HC-CDR3 comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 77-86; or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR regions;and ii) a light chain variable domain comprising an LC-CDR1 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 90-99, an LC-CDR2 comprising (and in some embodimentsconsisting of) the amino acid sequence of any one of SEQ ID NOs:100-109, and an LC-CDR3 comprising (and in some embodiments consistingof) the amino acid sequence of any one of SEQ ID NOs: 110-119; or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions in the LC-CDR regions.

In some embodiments, the anti-AMC construct comprises an antibody moietythat specifically binds to a complex comprising an AFP peptide and anMHC class I protein, wherein the antibody moiety comprises a) a heavychain variable domain comprising (and in some embodiments consisting of)the amino acid sequence of any one of SEQ ID NOs: 17-26 or a variantthereof having at least about 95% (such as at least about any of 95%,96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs:17-26; and b) a light chain variable domain comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 27-36 or a variant thereof having at least about 95% (such as atleast about any of 95%, 96%, 97%, 98%, or 99%) sequence identity to anyone of SEQ ID NOs: 27-36. In some embodiments, the antibody moietycomprises a heavy chain variable domain comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 17-26 and a light chain variable domain comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 27-36.

In some embodiments, the anti-AMC construct comprises a first antibodymoiety that competes for binding to a target AFP/MHC class I complexwith a second antibody moiety according to any of the antibody moietiesdescribed above. In some embodiments, the first antibody moiety binds tothe same, or substantially the same, epitope as the second antibodymoiety. In some embodiments, binding of the first antibody moiety to thetarget AFP/MHC class I complex inhibits binding of the second antibodymoiety to the target AFP/MHC class I complex by at least about 70% (suchas by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or 99%), orvice versa. In some embodiments, the first antibody moiety and thesecond antibody moiety cross-compete for binding to the target AFP/MHCclass I complex, i.e., each of the first and second antibody moietiescompetes with the other for binding to the target AFP/MHC class Icomplex.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the isolated anti-AMCconstruct is a full-length antibody. In some embodiments, the isolatedanti-AMC construct is monospecific. In some embodiments, the isolatedanti-AMC construct is multi-specific. In some embodiments, the isolatedanti-AMC construct is bispecific. In some embodiments, the isolatedanti-AMC molecule is a tandem scFv, a diabody (Db), a single chaindiabody (scDb), a dual-affinity retargeting (DART) antibody, a dualvariable domain (DVD) antibody, a knob-into-hole (KiH) antibody, a dockand lock (DNL) antibody, a chemically cross-linked antibody, aheteromultimeric antibody, or a heteroconjugate antibody. In someembodiments, the isolated anti-AMC construct is a tandem scFv comprisingtwo scFvs linked by a peptide linker. In some embodiments, the peptidelinker comprises (and in some embodiments consists of) the amino acidsequence GGGGS.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theisolated anti-AMC construct further comprises a second antigen-bindingmoiety that specifically binds to a second antigen. In some embodiments,the second antigen-binding moiety is an antibody moiety. In someembodiments, the second antigen is an antigen on the surface of a Tcell. In some embodiments, the T cell is selected from the groupconsisting of a cytotoxic T cell, a helper T cell, and a natural killerT cell. In some embodiments, the second antigen is selected from thegroup consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28, OX40, GITR, CD137,CD27, CD40L, and HVEM. In some embodiments, the second antigen is CD3ε,and the isolated anti-AMC construct is a tandem scFv comprising anN-terminal scFv specific for the AFP/MHC class I complex and aC-terminal scFv specific for CD3ε. In some embodiments, the secondantigen is an antigen on the surface of a natural killer cell, aneutrophil, a monocyte, a macrophage, or a dendritic cell.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theisolated anti-AMC construct is a chimeric antigen receptor. In someembodiments, the chimeric antigen receptor comprises an extracellulardomain comprising the antibody moiety, a transmembrane domain, and anintracellular signaling domain. In some embodiments, the intracellularsignaling domain comprises a CD3ζ intracellular signaling sequence and aco-stimulatory signaling sequence. In some embodiments, theco-stimulatory signaling sequence is a CD28 intracellular signalingsequence.

In some embodiments, according to any of the anti-AMC constructs (suchas isolated anti-AMC constructs) described above, the anti-AMC constructcomprises an antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theisolated anti-AMC construct is an immunoconjugate comprising theantibody moiety and an effector molecule. In some embodiments, theeffector molecule is a therapeutic agent selected from the groupconsisting of a drug, a toxin, a radioisotope, a protein, a peptide, anda nucleic acid. In some embodiments, the therapeutic agent is a drug ora toxin. In some embodiments, the effector molecule is a label.

In yet other embodiments, there is provided a pharmaceutical compositioncomprising an anti-AMC construct (such as an isolated anti-AMCconstruct) according to any of the embodiments described above. In someembodiments, the pharmaceutical composition further comprises a cell(such as an effector cell) associated with the anti-AMC construct. Insome embodiments, there is provided a host cell expressing or associatedwith an anti-AMC construct or polypeptide component thereof. In someembodiments, there is provided a nucleic acid encoding an anti-AMCconstruct or polypeptide component thereof. In some embodiments, thereis provided a vector comprising the nucleic acid. In some embodiments,there is provided an effector cell expressing or associated with ananti-AMC construct. In some embodiments, the effector cell is a T cell.

In some embodiments, there is provided a method of detecting a cellpresenting a complex comprising an AFP peptide and an MHC class Iprotein on its surface, comprising contacting the cell with an anti-AMCconstruct (such as an isolated anti-AMC construct) according to any ofthe embodiments described above comprising a) an antibody moiety thatspecifically binds to a complex comprising the AFP peptide and the MHCclass I protein and b) a label, and detecting the presence of the labelon the cell.

In some embodiments, there is provided a method of treating anindividual having an AFP-positive disease, comprising administering tothe individual an effective amount of a pharmaceutical compositioncomprising an anti-AMC construct (such as an isolated anti-AMCconstruct) according to any of the embodiments described above. In someembodiments, the pharmaceutical composition further comprises a cell(such as an effector cell) associated with the isolated anti-AMCconstruct. In some embodiments, there is provided a method of treatingan individual having an AFP-positive disease, comprising administeringto the individual an effective amount of an effector cell expressing anyof the anti-AMC CARs described above. In some embodiments, the effectorcell is a T cell. In some embodiments, the administration is to aninjection site distal to a first disease site in the individual. In someembodiments, the injection site is a first tumor distal to the firstdisease site. In some embodiments, the first disease site is anAFP-positive tumor. In some embodiments, the AFP-positive disease iscancer. In some embodiments, the cancer is hepatocellular carcinoma orgerm cell tumor. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand metastasis is inhibited. In some embodiments, the cancer ismetastatic hepatocellular carcinoma.

In some embodiments, there is provided a method of diagnosing anindividual having an AFP-positive disease, comprising: a) administeringan effective amount of an isolated anti-AMC construct according to anyof the embodiments described above to the individual; and b) determiningthe level of the label in the individual, wherein a level of the labelabove a threshold level indicates that the individual has theAFP-positive disease. In some embodiments, there is provided a method ofdiagnosing an individual having an AFP-positive disease, comprising: a)contacting a sample derived from the individual with an isolatedanti-AMC construct according to any of the embodiments described above;and b) determining the number of cells bound with the isolated anti-AMCconstruct in the sample, wherein a value for the number of cells boundwith the isolated anti-AMC construct above a threshold level indicatesthat the individual has the AFP-positive disease. In some embodiments,the AFP-positive disease is cancer. In some embodiments, the cancer ishepatocellular carcinoma or germ cell tumor. In some embodiments, thecancer is hepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma and metastasis is inhibited. In someembodiments, the cancer is metastatic hepatocellular carcinoma.

Also provided are methods of making any of the constructs describedherein, articles of manufacture, and kits that are suitable for themethods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the size exclusion chromatography (SEC) chromatogram ofAFP158 peptide/HLA-A*02:01 complex following concentration byultrafiltration. Properly folded peptide/MHC complex monomers: 212.49mL; misfolded aggregates: 111.27 mL; free 132M: 267.21 mL.

FIG. 2 shows reducing SDS-PAGE analysis to determine the purity ofAFP158/HLA-A*02:01 complex isolated following SEC. Major bandscorrespond to HLA-A*02:01 and (32M subunits.

FIG. 3 shows the results of phage clone ELISA for specific binding ofbiotinylated AFP158 peptide/HLA-A*02:01 (Bio-AFP158) versus biotinylatedcontrol peptide/HLA-A*02:01 (Bio-control).

FIG. 4 shows the results of phage clone FACS binding assays for bindingof (32M-loaded, β2M/AFP158 peptide-loaded and β2M/hTERT peptide-loadedT2 cells.

FIG. 5 shows the results of phage clone FACS binding assays forcross-reactivity with β2M/human AFP158 peptide-loaded T2 cells andβ2M/mouse AFP158 peptide-loaded T2 cells versus β2M-loaded T2 cells.

FIG. 6 shows the results of phage clone #52 FACS binding assays for T2cells loaded with AFP158 peptide having single alanine substitutions atvarying positions.

FIG. 7 shows the results of phage clone FACS binding assays for bindingof β2M AFP158 peptide-loaded T2 cells, T2 cells loaded with β2M and amixture of peptides derived from normally expressed endogenous proteinsor β2M-loaded T2 cells.

FIG. 8 shows SDS-PAGE analysis for molecular weight determination ofpurified anti-AFP158/MHC bispecific antibodies.

FIG. 9 shows SEC chromatograms of exemplary purified AFP158 bispecificantibodies to assess level of aggregation. anti-AFP158/MHC bispecificantibody monomer: ˜15.8 mL.

FIG. 10 shows the T-cell killing of multiple cancer cell lines mediatedby anti-AFP158/MHC bispecific antibodies (BsAb) at varyingconcentrations. For each concentration, the bars from left to rightrepresent HEPG2, SK-HEP1, Hela, HEP3B, Raji, Jurkat, Daudi, and K562cells lines.

FIG. 11 shows a schematic representation of a chimeric antigen receptorconstruct.

FIG. 12 shows the killing of HEPG2, SK-HEP1, and SK-HEP1-MiniG celllines mediated by T cells expressing a panel of anti-AFP158/MHC CARs.

FIG. 13 shows the killing of a panel of cancer cell lines positive ornegative for AFP158 and HLA-A*02:01, mediated by T cells expressing anexemplary anti-AFP158/MHC CAR. The tissue of origin of each cell line,the AFP/AFP158 peptide expression and whether the cells express theHLA-A02 allele are indicated in the table.

FIG. 14A shows the release of IL-2, IL-4, IL-6, and IL-8 afterco-incubation of AFP158 CAR transduced T cells or mock-transduced Tcells with cancer cell lines positive or negative for AFP158 andHLA-A*02:01.

FIG. 14B shows the release of IL-10, GM-CSF, IFN-γ, and TNF-α afterco-incubation of AFP158 CAR transduced T cells or mock-transduced Tcells with cancer cell lines positive or negative for AFP158 andHLA-A*02:01.

FIG. 15A shows tumor growth in HepG2 subcutaneous xenograft mice treatedwith intravenous injection of mock-transduced T cells or AFP158CAR-transduced T cells, or left untreated.

FIG. 15B shows tumor growth in HepG2 subcutaneous xenograft mice treatedwith intratumoral injection of mock-transduced T cells or AFP158CAR-transduced T cells, or left untreated.

FIG. 16A shows the change in the photon emission from luciferase-taggedHepG2 (HepG2-Luc2) intraperitoneal xenograft mice at day 70 aftertreatment with intraperitoneal injection of mock-transduced T cells orAFP158 CAR-transduced T cells, or no treatment.

FIG. 16B shows photon emission images of the HepG2-Luc2 tumor-bearingmice at day 70.

FIG. 17 shows tumor growth in SK-Hep1-MiniG subcutaneous xenograft micetreated with intravenous or intratumoral injection of AFP158CAR-transduced T cells or intravenous injection of mock-transduced Tcells.

FIG. 18 shows SDS-PAGE analysis to determine the purity of full-lengthanti-AFP158/MHC mouse chimeric IgG1 antibodies.

FIG. 19 shows FACS analysis of full-length anti-AFP158/MHC mousechimeric IgG1 (black line) or negative control (secondary antibodyalone, gray line) binding to SK-HEP1-miniG cells presentingAFP158/HLA-A*02:01.

FIG. 20 shows the results of ELISA for full-length anti-AFP158/MHC mousechimeric IgG1 binding to AFP158/HLA-A*02:01 complex, recombinant AFPprotein or free AFP158 peptide. Left panel: dose dependence curve offull-length anti-AFP158/MHC mouse chimeric IgG1; Right: OD450 ofantibody binding at 100 ng/mL for AFP158 peptide/MHC (MHC), AFP protein(AFP) and AFP158 peptide (Peptide).

FIG. 21 shows body weight measurements over time (up to 35 days post1^(st) dose) for mice injected intratumorally with AFP158 CAR-T cells orcontrols.

FIG. 22 shows tumor growth kinetics in bilateral SK-Hep1-MiniGsubcutaneous xenograft mice treated with intravenous or intratumoralinjection of AFP158 CAR-transduced T cells, intratumoral injection ofAFP158 CAR-transduced T cells in combination with APCs, or intratumoralinjection of mock-transduced T cells. Intratumoral injections wereinjected into right flank tumors.

FIG. 23 shows immunohistochemical staining of human CD3 in tumorsections from bilateral SK-Hep1-MiniG subcutaneous xenograft micetreated with intratumoral injection into right flank tumors of AFP158CAR-transduced T cells in combination with APCs. L, left tumor; R, righttumor.

FIG. 24 shows flow cytometry analysis of AFP158 CAR-transducedperipheral blood lymphocytes; cells were stained with AFP158/HLA-A*02:01tetramers and co-stained with anti-CD3, antibody, anti-CD4 antibody, oranti-CD8 antibody. Mock-transduced cells and cells without staining wereincluded as controls.

FIG. 25 shows flow cytometry analysis of the degranulation of AFP158CAR-transduced T cells after co-culturing with target cells (HepG2,SK-HEP-1 or SK-Hep1-MiniG); transduced T-cells were gated for CAR andCD8 expression and stained for anti-CD107a.

FIG. 26 shows CAR cell surface distribution in cells transduced toexpress a representative AFP158 CAR; cells were stained withAFP158/HLA-A*02:01 tetramer-PE.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides isolated constructs (referred to hereinas “anti-AMC constructs”) that comprise an antibody moiety (referred toherein as an “anti-AMC antibody moiety”) that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein (referredto herein as an “AFP/MHC class I complex,” or “AMC”). The anti-AMCconstructs specifically recognize AFP/MHC class I complexes (such asMHC-presented AFP peptides on the surface of cells expressing AFP), asopposed to circulating AFP protein or free AFP peptides. When armed asanti-CD3 bispecific antibodies or present in a chimeric antigen receptor(CAR) expressed by a T cell, the anti-AMC antibody moiety specificallyredirected human T cells to kill AMC-presenting target cells, such asAMC-presenting cancer cells. This strategy provides a significanttechnical advantage over using antibodies directed against the AFPprotein, which cannot specifically target AMC-presenting cells (i.e.,cells presenting on their surface an AFP peptide bound to an MHC class Imolecule). Furthermore, when fused to a detectable moiety, the anti-AMCantibody moiety allows for diagnosis and prognosis of AFP-positivediseases or disorders with high sensitivity to changes in the number anddistribution of AMC-presenting cells, a potentially more relevantmeasure of disease progression than circulating AFP levels.

Using phage display technology, we generated multiple monoclonalantibodies that are specific and high affinity against human AFP158peptide/HLA-A*02:01 complex, as well as AFP158/MHC complexes formed inthe context of other subtypes of the HLA-A02 allele. Flow cytometry andT-cell mediated cytotoxicity assays demonstrated that the antibodiesrecognized AFP peptide-pulsed T2 cells and AMC-presenting cancer celllines, in an AFP- and HLA-A*02:01-restricted manner. When armed asanti-CD3 bispecific antibodies or CAR T cells, the antibodiesre-directed human T cells to kill AFP-positive and HLA-A*02:01-positivetarget cancer cells. The data presented herein demonstrate thatantibodies against a secreted cancer antigen in the context of an HLAcomplex can be effective therapeutic agents for cancer indications, suchas solid tumor indications.

The present application thus provides constructs (such as isolatedconstructs) comprising an antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein. Theconstruct can be, for example, a full-length anti-AMC antibody, amulti-specific anti-AMC molecule (such as a bispecific anti-AMCantibody), an anti-AMC chimeric antigen receptor (“CAR”), or an anti-AMCimmunoconjugate.

In another aspect, there are provided nucleic acids encoding theanti-AMC constructs or the anti-AMC antibody moiety portion of theconstructs.

In another aspect, there are provided compositions comprising ananti-AMC construct comprising an antibody moiety that specifically bindsto a complex comprising an AFP-peptide and an MHC class I protein. Thecomposition can be a pharmaceutical composition comprising an anti-AMCconstruct or an effector cell expressing or associated with the anti-AMCconstruct (for example a T cell expressing an anti-AMC CAR).

Also provided are methods of making and using the anti-AMC constructs(or cells expressing or associated with the anti-AMC constructs) fortreatment or diagnostic purposes, as well as kits and articles ofmanufacture useful for such methods.

Definitions

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results, including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the recurrence of thedisease, delay or slowing the progression of the disease, amelioratingthe disease state, providing a remission (partial or total) of thedisease, decreasing the dose of one or more other medications requiredto treat the disease, delaying the progression of the disease,increasing or improving the quality of life, increasing weight gain,and/or prolonging survival. Also encompassed by “treatment” is areduction of pathological consequence of cancer (such as, for example,tumor volume). The methods of the invention contemplate any one or moreof these aspects of treatment.

The terms “recurrence,” “relapse” or “relapsed” refers to the return ofa cancer or disease after clinical assessment of the disappearance ofdisease. A diagnosis of distant metastasis or local recurrence can beconsidered a relapse.

The term “refractory” or “resistant” refers to a cancer or disease thathas not responded to treatment.

“Activation”, as used herein in relation to T cells, refers to the stateof a T cell that has been sufficiently stimulated to induce detectablecellular proliferation. Activation can also be associated with inducedcytokine production, and detectable effector functions.

The term “antibody moiety” includes full-length antibodies andantigen-binding fragments thereof. A full-length antibody comprises twoheavy chains and two light chains. The variable regions of the light andheavy chains are responsible for antigen binding. The variables regionin both chains generally contain three highly variable loops called thecomplementarity determining regions (CDRs) (light chain (LC) CDRsincluding LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs includingHC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies andantigen-binding fragments disclosed herein may be defined or identifiedby the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997;Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Thethree CDRs of the heavy or light chains are interposed between flankingstretches known as framework regions (FRs), which are more highlyconserved than the CDRs and form a scaffold to support the hypervariableloops. The constant regions of the heavy and light chains are notinvolved in antigen binding, but exhibit various effector functions.Antibodies are assigned to classes based on the amino acid sequence ofthe constant region of their heavy chain. The five major classes orisotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which arecharacterized by the presence of α, δ, ε, γ, and μ. heavy chains,respectively. Several of the major antibody classes are divided intosubclasses such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3(γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2(α2 heavy chain).

The term “antigen-binding fragment” as used herein refers to an antibodyfragment including, for example, a diabody, a Fab, a Fab′, a F(ab′)2, anFv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, abispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), an scFv dimer(bivalent diabody), a multispecific antibody formed from a portion of anantibody comprising one or more CDRs, a camelized single domainantibody, a nanobody, a domain antibody, a bivalent domain antibody, orany other antibody fragment that binds to an antigen but does notcomprise a complete antibody structure. An antigen-binding fragment iscapable of binding to the same antigen to which the parent antibody or aparent antibody fragment (e.g., a parent scFv) binds. In someembodiments, an antigen-binding fragment may comprise one or more CDRsfrom a particular human antibody grafted to a framework region from oneor more different human antibodies.

The term “epitope” as used herein refers to the specific group of atomsor amino acids on an antigen to which an antibody or antibody moietybinds. Two antibodies or antibody moieties may bind the same epitopewithin an antigen if they exhibit competitive binding for the antigen.

As used herein, a first antibody moiety “competes” for binding to atarget AMC with a second antibody moiety when the first antibody moietyinhibits target AMC binding of the second antibody moiety by at leastabout 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentrationof the first antibody moiety, or vice versa. A high throughput processfor “binning” antibodies based upon their cross-competition is describedin PCT Publication No. WO 03/48731.

As use herein, the term “specifically binds” or “is specific for” refersto measurable and reproducible interactions, such as binding between atarget and an antibody or antibody moiety, that is determinative of thepresence of the target in the presence of a heterogeneous population ofmolecules, including biological molecules. For example, an antibody orantibody moiety that specifically binds to a target (which can be anepitope) is an antibody or antibody moiety that binds this target withgreater affinity, avidity, more readily, and/or with greater durationthan its bindings to other targets. In some embodiments, an antibody orantibody moiety that specifically binds to an antigen reacts with one ormore antigenic determinants of the antigen (for example an AFPpeptide/MHC class I protein complex) with a binding affinity that is atleast about 10 times its binding affinity for other targets.

An “isolated” anti-AMC construct as used herein refers to an anti-AMCconstruct that (1) is not associated with proteins found in nature, (2)is free of other proteins from the same source, (3) is expressed by acell from a different species, or, (4) does not occur in nature.

The term “isolated nucleic acid” as used herein is intended to mean anucleic acid of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated nucleic acid” (1)is not associated with all or a portion of a polynucleotide in which the“isolated nucleic acid” is found in nature, (2) is operably linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.These particular regions have been described by Kabat et al., J. Biol.Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and HumanServices, “Sequences of proteins of immunological interest” (1991); byChothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or grafted antibodies or variants thereof is intended tobe within the scope of the term as defined and used herein. The aminoacid residues which encompass the CDRs as defined by each of the abovecited references are set forth below in Table 1 as a comparison.

TABLE 1 CDR DEFINITIONS Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

The term “chimeric antibodies” refer to antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit a biological activity of thisinvention (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The term “semi-synthetic” in reference to an antibody or antibody moietymeans that the antibody or antibody moiety has one or more naturallyoccurring sequences and one or more non-naturally occurring (i.e.,synthetic) sequences.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the heavy and light chain)that contribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. In some embodiments, the scFvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the scFv to form the desired structure forantigen binding. For a review of scFv, see Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing scFv fragments (see preceding paragraph) typically withshort linkers (such as about 5 to about 10 residues) between the V_(H)and V_(L) domains such that inter-chain but not intra-chain pairing ofthe V domains is achieved, resulting in a bivalent fragment, i.e.,fragment having two antigen-binding sites. Bispecific diabodies areheterodimers of two “crossover” scFv fragments in which the V_(H) andV_(L) domains of the two antibodies are present on different polypeptidechains. Diabodies are described more fully in, for example, EP 404,097;WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (HVR) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Percent (%) amino acid sequence identity” or “homology” with respect tothe polypeptide and antibody sequences identified herein is defined asthe percentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the polypeptide beingcompared, after aligning the sequences considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,Megalign (DNASTAR), or MUSCLE software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program MUSCLE (Edgar, R. C., Nucleic Acids Research32(5):1792-1797, 2004; Edgar, R. C., BMC Bioinformatics 5(1):113, 2004).

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In some embodiments, an FcR ofthis invention is one that binds an IgG antibody (a γ receptor) andincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain (see review M. in Daëron, Annu. Rev.Immunol. 15:203-234 (1997)). The term includes allotypes, such asFcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Va1158, FcγRIIA-R131and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn isstructurally similar to major histocompatibility complex (MHC) andconsists of an α-chain noncovalently bound to β2-microglobulin. Themultiple functions of the neonatal Fc receptor FcRn are reviewed inGhetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays arole in the passive delivery of immunoglobulin IgGs from mother to youngand the regulation of serum IgG levels. FcRn can act as a salvagereceptor, binding and transporting pinocytosed IgGs in intact form bothwithin and across cells, and rescuing them from a default degradativepathway.

The “CH1 domain” of a human IgG Fc region (also referred to as “C1” of“H1” domain) usually extends from about amino acid 118 to about aminoacid 215 (EU numbering system).

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S-S bondsin the same positions.

The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of“H2” domain) usually extends from about amino acid 231 to about aminoacid 340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec Immunol. 22:161-206 (1985).

The “CH3 domain” (also referred to as “C2” or “H3” domain) comprises thestretch of residues C-terminal to a CH2 domain in an Fc region (i.e.from about amino acid residue 341 to the C-terminal end of an antibodysequence, typically at amino acid residue 446 or 447 of an IgG).

A “functional Fc fragment” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art.

An antibody with a variant IgG Fc with “altered” FcR binding affinity orADCC activity is one which has either enhanced or diminished FcR bindingactivity (e.g., FcγR or FcRn) and/or ADCC activity compared to a parentpolypeptide or to a polypeptide comprising a native sequence Fc region.The variant Fc which “exhibits increased binding” to an FcR binds atleast one FcR with higher affinity (e.g., lower apparent K_(d) or IC₅₀value) than the parent polypeptide or a native sequence IgG Fc.According to some embodiments, the improvement in binding compared to aparent polypeptide is about 3 fold, such as about any of 5, 10, 25, 50,60, 100, 150, 200, or up to 500 fold, or about 25% to 1000% improvementin binding. The polypeptide variant which “exhibits decreased binding”to an FcR, binds at least one FcR with lower affinity (e.g., higherapparent K_(d) or higher IC₅₀ value) than a parent polypeptide. Thedecrease in binding compared to a parent polypeptide may be about 40% ormore decrease in binding.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1998).

The polypeptide comprising a variant Fc region which “exhibits increasedADCC” or mediates antibody-dependent cell-mediated cytotoxicity (ADCC)in the presence of human effector cells more effectively than apolypeptide having wild type IgG Fc or a parent polypeptide is one whichin vitro or in vivo is substantially more effective at mediating ADCC,when the amounts of polypeptide with variant Fc region and thepolypeptide with wild type Fc region (or the parent polypeptide) in theassay are essentially the same. Generally, such variants will beidentified using any in vitro ADCC assay known in the art, such asassays or methods for determining ADCC activity, e.g. in an animal modeletc. In some embodiments, the variant is from about 5 fold to about 100fold, e.g. from about 25 to about 50 fold, more effective at mediatingADCC than the wild type Fc (or parent polypeptide).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed. Polypeptide variantswith altered Fc region amino acid sequences and increased or decreasedC1q binding capability are described in U.S. Pat. No. 6,194,551B1 andWO99/51642. The contents of those patent publications are specificallyincorporated herein by reference. See, also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared times 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

An “effective amount” of an anti-AMC construct or composition asdisclosed herein, is an amount sufficient to carry out a specificallystated purpose. An “effective amount” can be determined empirically andby known methods relating to the stated purpose.

The term “therapeutically effective amount” refers to an amount of ananti-AMC construct or composition as disclosed herein, effective to“treat” a disease or disorder in an individual. In the case of cancer,the therapeutically effective amount of the anti-AMC construct orcomposition as disclosed herein can reduce the number of cancer cells;reduce the tumor size or weight; inhibit (i.e., slow to some extent andpreferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent the anti-AMC construct or composition as disclosed herein canprevent growth and/or kill existing cancer cells, it can be cytostaticand/or cytotoxic. In some embodiments, the therapeutically effectiveamount is a growth inhibitory amount. In some embodiments, thetherapeutically effective amount is an amount that extends the survivalof a patient. In some embodiments, the therapeutically effective amountis an amount that improves progression free survival of a patient.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

The term “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to theanti-AMC antibody moiety. The label may be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Anti-AMC Constructs

In one aspect, the present invention provides AFP/MHC class Icomplex-specific constructs (anti-AMC constructs) that comprise anantibody moiety that specifically binds to a complex comprising an AFPpeptide and an MHC class I protein (“AFP/MHC class I complex,” or“AMC”). The specificity of the anti-AMC construct derives from ananti-AMC antibody moiety, such as a full-length antibody orantigen-binding fragment thereof, that specifically binds to the AMC. Insome embodiments, reference to a moiety (such as an antibody moiety)that specifically binds to a complex comprising an AFP peptide and anMHC class I protein means that the moiety binds to the AMC with a) anaffinity that is at least about 10 (including for example at least aboutany of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 ormore) times its binding affinity for each of full-length AFP, free AFPpeptide, MHC class I protein not bound to a peptide, and MHC class Iprotein bound to a non-AFP peptide; or b) a K_(d) no more than about1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50,1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) timesits K_(d) for binding to each of full-length AFP, free AFP peptide, MHCclass I protein not bound to a peptide, and MHC class I protein bound toa non-AFP peptide. Binding affinity can be determined by methods knownin the art, such as ELISA, fluorescence activated cell sorting (FACS)analysis, or radioimmunoprecipitation assay (RIA). K_(d) can bedetermined by methods known in the art, such as surface plasmonresonance (SPR) assay utilizing, for example, Biacore instruments, orkinetic exclusion assay (KinExA) utilizing, for example, Sapidyneinstruments.

Contemplated anti-AMC constructs include, for example, full-lengthanti-AMC antibodies, multi-specific (such as bispecific) anti-AMCmolecules, anti-AMC chimeric antigen receptors (CARs), and anti-AMCimmunoconjugates.

For example, in some embodiments, there is provided an anti-AMCconstruct (such as an isolated anti-AMC construct) comprising ananti-AMC antibody moiety that specifically binds to a complex comprisingan AFP peptide and an MHC class I protein. In some embodiments, the AFPpeptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC class Iprotein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01 (GenBank Accession No.: AA020853). In some embodiments, theanti-AMC construct is non-naturally occurring. In some embodiments, theanti-AMC construct is a full-length antibody. In some embodiments, theanti-AMC construct is a multi-specific (such as bispecific) molecule. Insome embodiments, the anti-AMC construct is a chimeric antigen receptor.In some embodiments, the anti-AMC construct is an immunoconjugate. Insome embodiments, the anti-AMC construct binds the AMC with a K_(d)between about 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM,including any ranges between these values). In some embodiments, theanti-AMC construct cross-reacts with at least one (such as at least anyof 2, 3, 4, 5, or 6) complex comprising the MHC class I protein and avariant of the AFP peptide having one amino acid substitution (such as aconservative amino acid substitution). In some embodiments, the anti-AMCconstruct cross-reacts with at least one (such as at least any of 2, 3,4, or 5) complex comprising the AFP peptide and a different subtype ofthe MHC class I protein.

In some embodiments, there is provided an anti-AMC construct comprisingan anti-AMC antibody moiety that specifically binds to a complexcomprising an AFP158 peptide (SEQ ID NO: 4) and HLA-A*02:01. In someembodiments, the anti-AMC construct is non-naturally occurring. In someembodiments, the anti-AMC construct is a full-length antibody. In someembodiments, the anti-AMC construct is a multi-specific (such asbispecific) molecule. In some embodiments, the anti-AMC construct is achimeric antigen receptor. In some embodiments, the anti-AMC constructis an immunoconjugate. In some embodiments, the anti-AMC construct bindsthe AMC with a K_(d) between about 0.1 pM to about 500 nM (such as aboutany of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM,100 nM, or 500 nM, including any ranges between these values). In someembodiments, the anti-AMC construct cross-reacts with at least one (suchas at least any of 2, 3, 4, 5, or 6) complex comprising the MHC class Iprotein and a variant of the AFP peptide having one amino acidsubstitution (such as a conservative amino acid substitution). In someembodiments, the anti-AMC construct cross-reacts with at least one (suchas at least any of 2, 3, 4, or 5) complex comprising the AFP peptide anda different subtype of the MHC class I protein.

In some embodiments, there is provided an anti-AMC construct comprisingan anti-AMC antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theanti-AMC antibody moiety comprises: i) a heavy chain variable domainsequence comprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid. In some embodiments, theanti-AMC construct is non-naturally occurring. In some embodiments, theanti-AMC construct is a full-length antibody. In some embodiments, theanti-AMC construct is a multi-specific (such as bispecific) molecule. Insome embodiments, the anti-AMC construct is a chimeric antigen receptor.In some embodiments, the anti-AMC construct is an immunoconjugate. Insome embodiments, the anti-AMC construct binds the AMC with a K_(d)between about 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM,including any ranges between these values). In some embodiments, theanti-AMC construct cross-reacts with at least one (such as at least anyof 2, 3, 4, 5, or 6) complex comprising the MHC class I protein and avariant of the AFP peptide having one amino acid substitution (such as aconservative amino acid substitution). In some embodiments, the anti-AMCconstruct cross-reacts with at least one (such as at least any of 2, 3,4, or 5) complex comprising the AFP peptide and a different subtype ofthe MHC class I protein.

In some embodiments, there is provided an anti-AMC construct comprisingan anti-AMC antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theanti-AMC antibody moiety comprises: i) a heavy chain variable domainsequence comprising an HC-CDR1 comprising (and in some embodimentsconsisting of) the amino acid sequence of any one of SEQ ID NOs: 57-66;or a variant thereof comprising up to about 5 (for example about any of1, 2, 3, 4, or 5) amino acid substitutions; an HC-CDR2 comprising (andin some embodiments consisting of) the amino acid sequence of any one ofSEQ ID NOs: 67-76; or a variant thereof comprising up to about 5 (forexample about any of 1, 2, 3, 4, or 5) amino acid substitutions; and anHC-CDR3 comprising (and in some embodiments consisting of) the aminoacid sequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)amino acid substitutions; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising (and in some embodiments consisting of)the amino acid sequence of any one of SEQ ID NOs: 90-99; or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising (and in someembodiments consisting of) the amino acid sequence of any one of SEQ IDNOs: 100-109; or a variant thereof comprising up to about 3 (for exampleabout any of 1, 2, or 3) amino acid substitutions; and an LC-CDR3comprising (and in some embodiments consisting of) the amino acidsequence of any one of SEQ ID NOs: 110-119; or a variant thereofcomprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)amino acid substitutions. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, theanti-AMC construct binds the AMC with a Kd between about 0.1 pM to about500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges betweenthese values). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, 5, or 6) complexcomprising the MHC class I protein and a variant of the AFP peptidehaving one amino acid substitution (such as a conservative amino acidsubstitution). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, or 5) complexcomprising the AFP peptide and a different subtype of the MHC class Iprotein.

In some embodiments, there is provided an anti-AMC construct comprisingan anti-AMC antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MHC class I protein, wherein theanti-AMC antibody moiety comprises: i) a heavy chain variable domainsequence comprising an HC-CDR1 comprising (and in some embodimentsconsisting of) the amino acid sequence of any one of SEQ ID NOs: 57-66;an HC-CDR2 comprising (and in some embodiments consisting of) the aminoacid sequence of any one of SEQ ID NOs: 67-76; and an HC-CDR3 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 77-86; or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions inthe HC-CDR sequences; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising (and in some embodiments consisting of)the amino acid sequence of any one of SEQ ID NOs: 90-99; an LC-CDR2comprising (and in some embodiments consisting of) the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 110-119; or a variant thereof comprising up to about5 (for example about any of 1, 2, 3, 4, or 5) amino acid substitutionsin the LC-CDR sequences. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, theanti-AMC construct binds the AMC with a Kd between about 0.1 pM to about500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges betweenthese values). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, 5, or 6) complexcomprising the MHC class I protein and a variant of the AFP peptidehaving one amino acid substitution (such as a conservative amino acidsubstitution). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, or 5) complexcomprising the AFP peptide and a different subtype of the MHC class Iprotein.

In some embodiments, there is provided an anti-AMC construct comprisingan anti-AMC antibody moiety that specifically binds to a complexcomprising an AFP peptide and an MCH class I protein, wherein theanti-AMC antibody moiety comprises a heavy chain variable domaincomprising (and in some embodiments consisting of) the amino acidsequence of any one of SEQ ID NOs: 17-26, or a variant thereof having atleast about 95% (for example at least about any of 96%, 97%, 98%, or99%) sequence identity, and a light chain variable domain comprising(and in some embodiments consisting of) the amino acid sequence of anyone of SEQ ID NOs: 27-36, or a variant thereof having at least about 95%(for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity. In some embodiments, the anti-AMC construct is non-naturallyoccurring. In some embodiments, the anti-AMC construct is a full-lengthantibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, theanti-AMC construct binds the AMC with a Kd between about 0.1 pM to about500 nM (such as about any of 0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM, including any ranges betweenthese values). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, 5, or 6) complexcomprising the MHC class I protein and a variant of the AFP peptidehaving one amino acid substitution (such as a conservative amino acidsubstitution). In some embodiments, the anti-AMC construct cross-reactswith at least one (such as at least any of 2, 3, 4, or 5) complexcomprising the AFP peptide and a different subtype of the MHC class Iprotein.

In some embodiments, there is provided an anti-AMC construct comprisinga first anti-AMC antibody moiety that competes for binding to a targetAFP/MHC class I complex with a second anti-AMC antibody moiety accordingto any of the anti-AMC antibody moieties described herein. In someembodiments, the first anti-AMC antibody moiety binds to the same, orsubstantially the same, epitope as the second anti-AMC antibody moiety.In some embodiments, binding of the first anti-AMC antibody moiety tothe target AFP/MHC class I complex inhibits binding of the secondanti-AMC antibody moiety to the target AFP/MHC class I complex by atleast about 70% (such as by at least about any of 75%, 80%, 85%, 90%,95%, 98% or 99%), or vice versa. In some embodiments, the first anti-AMCantibody moiety and the second anti-AMC antibody moiety cross-competefor binding to the target AFP/MHC class I complex, i.e., each of thefirst and second antibody moieties competes with the other for bindingto the target AFP/MHC class I complex.

The different aspects are discussed in various sections below in furtherdetail.

Anti-AMC Antibody Moiety

The anti-AMC constructs comprise an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein.

In some embodiments, the anti-AMC antibody moiety specifically binds toan AMC present on the surface of a cell. In some embodiments, the cellpresents on its surface abnormally high levels of AFP. In someembodiments, the cell is a cancer cell. In some embodiments, the cancercell is in a solid tumor. In some embodiments, the cancer cell is ametastatic cancer cell.

In some embodiments, the AFP peptide is an MHC class I-restrictedpeptide. In some embodiments, the AFP peptide is from about 8 to about12 (such as about any of 8, 9, 10, 11, or 12) amino acids in length. Insome embodiments, the AFP peptide is derived from human AFP (hAFP),mouse AFP (mAFP), or rat AFP (rAFP).

In some embodiments, the AFP peptide is derived from hAFP. In someembodiments, the AFP peptide comprises (and in some embodiments consistsof) the sequence of amino acids 137-145 of hAFP (PLFQVPEPV, SEQ ID NO:3), amino acids 158-166 of hAFP (FMNKFIYEI, SEQ ID NO: 4, also referredto herein as “AFP158”), amino acids 325-334 of hAFP (GLSPNLNRFL, SEQ IDNO: 5), or amino acids 542-50 of hAFP (GVALQTMKQ, SEQ ID NO: 6).

In some embodiments, the AFP peptide is derived from mAFP. In someembodiments, the AFP peptide comprises the sequence of amino acids154-162 of mAFP (FMNRFIYEV, SEQ ID NO: 16).

In some embodiments, the MHC class I protein is HLA-A, HLA-B, HLA-C,HLA-E, HLA-F, or HLA-G. In some embodiments, the MHC class I protein isHLA-A. In some embodiments, the HLA-A is HLA-A02. In some embodiments,the HLA-A02 is HLA-A*02:01.

In some embodiments, the anti-AMC antibody moiety is a full-lengthantibody. In some embodiments, the anti-AMC antibody moiety is anantigen-binding fragment, for example an antigen-binding fragmentselected from the group consisting of a Fab, a Fab′, a F(ab′)2, an Fvfragment, a disulfide stabilized Fv fragment (dsFv), and a single-chainantibody molecule (scFv). In some embodiments, the anti-AMC antibodymoiety is an scFv. In some embodiments, the anti-AMC antibody moiety ishuman, humanized, or semi-synthetic.

In some embodiments, the anti-AMC antibody moiety specifically binds tothe N-terminal portion of the AFP peptide in the complex. In someembodiments, the anti-AMC antibody moiety specifically binds to theC-terminal portion of the AFP peptide in the complex. In someembodiments, the anti-AMC antibody moiety specifically binds to themiddle portion of the AFP peptide in the complex.

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising an AFP peptide and an MHC class I protein, whereinthe anti-AMC antibody moiety cross-reacts with at least one complexcomprising the AFP peptide and an allelic variant of the MHC class Iprotein. In some embodiments, the allelic variant has up to about 10(such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acidsubstitutions when compared to the MHC class I protein. In someembodiments, the allelic variant is the same serotype as the MHC class Iprotein. In some embodiments, the allelic variant is a differentserotype than the MHC class I protein. In some embodiments, the anti-AMCantibody moiety does not cross-react with a complex comprising the AFPpeptide and any allelic variant of the MHC class I protein.

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising an AFP peptide and an MHC class I protein, whereinthe anti-AMC antibody moiety cross-reacts with at least one complexcomprising the MHC class I protein and a variant of the AFP peptidehaving one amino acid substitution (such as a conservativesubstitution). In some embodiments, the anti-AMC antibody moiety doesnot cross-react with a complex comprising the MHC class I protein andany variant of the AFP peptide.

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising an AFP peptide and an MHC class I protein, whereinthe anti-AMC antibody moiety cross-reacts with at least one complexcomprising the MHC class I protein and an interspecies variant of theAFP peptide. In some embodiments, for example, the AFP peptide is humanAFP peptide and the interspecies variant of the AFP peptide is a mouseor rat variant thereof. In some embodiments, the anti-AMC antibodymoiety does not cross-react with a complex comprising the MHC class Iprotein and any interspecies variant of the AFP peptide.

In some embodiments, the anti-AMC antibody moiety (or the anti-AMCconstruct comprising the anti-AMC antibody moiety) binds to the complexcomprising the AFP peptide and the MHC class I protein with an affinitythat is at least about 10 (including for example at least about any of10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more)times its binding affinity for each of full-length AFP, free AFPpeptide, MHC class I protein not bound to a peptide, and MHC class Iprotein bound to a non-AFP peptide. In some embodiments, the anti-AMCantibody moiety (or the anti-AMC construct comprising the anti-AMCantibody moiety) binds to the complex comprising the AFP peptide and theMHC class I protein with a K_(d) no more than about 1/10 (such as nomore than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200,1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its K_(d) for bindingto each of full-length AFP, free AFP peptide, MHC class I protein notbound to a peptide, and MHC class I protein bound to a non-AFP peptide.

In some embodiments, the anti-AMC antibody moiety (or the anti-AMCconstruct comprising the anti-AMC antibody moiety) binds to the complexcomprising the AFP peptide and the MHC class I protein with a Kd betweenabout 0.1 pM to about 500 nM (such as about any of 0.1 pM, 1.0 pM, 10pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500 nM,including any ranges between these values). In some embodiments, theanti-AMC antibody moiety (or the anti-AMC construct comprising theanti-AMC antibody moiety) binds to the complex comprising the AFPpeptide and the MHC class I protein with a Kd between about 1 pM toabout 250 pM (such as about any of 1, 10, 25, 50, 75, 100, 150, 200, or250 pM, including any ranges between these values). In some embodiments,the anti-AMC antibody moiety (or the anti-AMC construct comprising theanti-AMC antibody moiety) binds to the complex comprising the AFPpeptide and the MHC class I protein with a Kd between about 1 nM toabout 500 nM (such as about any of 1, 10, 25, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, or 500 nM, including any ranges between thesevalues).

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising AFP158 (SEQ ID NO: 4) and an MHC class I protein(such as HLA-A02, for example HLA-A*02:01). In some embodiments, theanti-AMC antibody moiety further binds to at least one (including atleast about any of 2, 3, 4, 5, 6, or 7) of: a complex comprising an AFPpeptide of SEQ ID NO: 7 and an MHC class I protein (such as HLA-A02, forexample HLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO:8 and an MHC class I protein (such as HLA-A02, for example HLA-A*02:01);a complex comprising an AFP peptide of SEQ ID NO: 9 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 10 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); a complex comprising an AFP peptideof SEQ ID NO: 11 and an MHC class I protein (such as HLA-A02, forexample HLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO:12 and an MHC class I protein (such as HLA-A02, for exampleHLA-A*02:01); and a complex comprising an AFP peptide of SEQ ID NO: 13and an MHC class I protein (such as HLA-A02, for example HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds to:a complex comprising an AFP peptide of SEQ ID NO: 4 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 10 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); a complex comprising an AFP peptideof SEQ ID NO: 11 and an MHC class I protein (such as HLA-A02, forexample HLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO:12 and an MHC class I protein (such as HLA-A02, for exampleHLA-A*02:01); and a complex comprising an AFP peptide of SEQ ID NO: 13and an MHC class I protein (such as HLA-A02, for example HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds to:a complex comprising an AFP peptide of SEQ ID NO: 4 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 7 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); and a complex comprising an AFPpeptide of SEQ ID NO: 8 and an MHC class I protein (such as HLA-A02, forexample HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds to:a complex comprising an AFP peptide of SEQ ID NO: 4 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 8 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); a complex comprising an AFP peptideof SEQ ID NO: 10 and an MHC class I protein (such as HLA-A02, forexample HLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO:11 and an MHC class I protein (such as HLA-A02, for exampleHLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO: 12 andan MHC class I protein (such as HLA-A02, for example HLA-A*02:01); and acomplex comprising an AFP peptide of SEQ ID NO: 13 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds to:a complex comprising an AFP peptide of SEQ ID NO: 4 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 7 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); and a complex comprising an AFPpeptide of SEQ ID NO: 13 and an MHC class I protein (such as HLA-A02,for example HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds to:a complex comprising an AFP peptide of SEQ ID NO: 4 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01); a complex comprisingan AFP peptide of SEQ ID NO: 7 and an MHC class I protein (such asHLA-A02, for example HLA-A*02:01); a complex comprising an AFP peptideof SEQ ID NO: 9 and an MHC class I protein (such as HLA-A02, for exampleHLA-A*02:01); a complex comprising an AFP peptide of SEQ ID NO: 11 andan MHC class I protein (such as HLA-A02, for example HLA-A*02:01); and acomplex comprising an AFP peptide of SEQ ID NO: 13 and an MHC class Iprotein (such as HLA-A02, for example HLA-A*02:01).

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising AFP158 (SEQ ID NO: 4) and HLA-A*02:01. In someembodiments, the anti-antibody moiety cross-reacts with at least one(including at least about any of 2, 3, 4, 5, or 6) of: a complexcomprising AFP158 (SEQ ID NO: 4) and HLA-A*02:02 (GenBank Accession No.:AFL91480), a complex comprising AFP158 (SEQ ID NO: 4) and HLA-A*02:03(GenBank Accession No.: AAA03604), a complex comprising AFP158 (SEQ IDNO: 4) and HLA-A*02:05 (GenBank Accession No.: AAA03603), a complexcomprising AFP158 (SEQ ID NO: 4) and HLA-A*02:06 (GenBank Accession No.:CCB78868), a complex comprising AFP158 (SEQ ID NO: 4) and HLA-A*02:07(GenBank Accession No.: ACR55712), and a complex comprising AFP158 (SEQID NO: 4) and HLA-A*02:11 (GenBank Accession No.: CAB56609). In someembodiments, the anti-AMC antibody moiety cross-reacts with each of acomplex comprising AFP158 (SEQ ID NO: 4) and HLA-A*02:02, a complexcomprising AFP158 (SEQ ID NO: 4) and HLA-A*02:03, and a complexcomprising AFP158 (SEQ ID NO: 4) and HLA-A*02:11. In some embodiments,the anti-AMC antibody moiety cross-reacts with each of a complexcomprising AFP158 (SEQ ID NO: 4) and HLA-A*02:02, a complex comprisingAFP158 (SEQ ID NO: 4) and HLA-A*02:05, a complex comprising AFP158 (SEQID NO: 4) and HLA-A*02:06, a complex comprising AFP158 (SEQ ID NO: 4)and HLA-A*02:07, and a complex comprising AFP158 (SEQ ID NO: 4) andHLA-A*02:11.

In some embodiments, the anti-AMC antibody moiety is a semi-syntheticantibody moiety comprising fully human sequences and one or moresynthetic regions. In some embodiments, the anti-AMC antibody moiety isa semi-synthetic antibody moiety comprising a fully human light chainvariable domain and a semi-synthetic heavy chain variable domaincomprising fully human FR1, HC-CDR1, FR2, HC-CDR2, FR3, and FR4 regionsand a synthetic HC-CDR3. In some embodiments, the semi-synthetic heavychain variable domain comprises a fully synthetic HC-CDR3 having asequence from about 5 to about 25 (such as about any of 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) aminoacids in length. In some embodiments, the semi-synthetic heavy chainvariable domain or the synthetic HC-CDR3 is obtained from asemi-synthetic library (such as a semi-synthetic human library)comprising fully synthetic HC-CDR3s having a sequence from about 5 toabout 25 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length,wherein each amino acid in the sequence is randomly selected from thestandard human amino acids, minus cysteine. In some embodiments, thesynthetic HC-CDR3 is from about 10 to about 19 (such as about any of 10,11, 12, 13, 14, 15, 16, 17, 18 or 19) amino acids in length.

The anti-AMC antibody moieties in some embodiments comprise specificsequences or certain variants of such sequences. In some embodiments,the amino acid substitutions in the variant sequences do notsubstantially reduce the ability of the anti-AMC antibody moiety to bindthe AMC. For example, alterations that do not substantially reduce AMCbinding affinity may be made. Alterations that substantially improve AMCbinding affinity or affect some other property, such as specificityand/or cross-reactivity with related variants of the AMC, are alsocontemplated.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR3 comprising the amino acidsequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variantthereof comprising up to about 3 (for example about any of 1, 2, or 3)amino acid substitutions; and ii) a light chain variable domaincomprising an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR3 comprising the amino acidsequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii) a lightchain variable domain comprising an LC-CDR3 comprising the amino acidsequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121); wherein X can be anyamino acid.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or avariant thereof comprising up to about 3 (for example about any of 1, 2,or 3) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or avariant thereof comprising up to about 3 (for example about any of 1, 2,or 3) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii) a light chain variabledomain comprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121); wherein X can be any amino acid.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), anHC-CDR2 comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQID NO: 88), and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); or a variant thereof comprisingup to about 3 (such as about any of 1, 2, or 3) amino acid substitutionsin the HC-CDR sequences; and ii) a light chain variable domaincomprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); or a variant thereof comprising up to about 3 (such as about anyof 1, 2, or 3) amino acid substitutions in the LC-CDR sequences; whereinX can be any amino acid.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), anHC-CDR2 comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQID NO: 88), and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii) a light chain variabledomain comprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid. The sequences of the CDRs notedherein are provided in Table 2 below.

TABLE 2  HC-CDR1 consensus SEQ ID NO: 87G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W HC-CDR2 consensus SEQ ID NO: 88I/S-K/S-X-H/Y-X-G-X-T HC-CDR3 consensus SEQ ID NO: 89A/G-X-W/Y-Y-X-X-X-F/Y-D LC-CDR1 consensus SEQ ID NO: 120S/T-G/S-D/N-IN-A/G-A/SN-X-H/Y LC-CDR3 consensus SEQ ID NO: 121Q-S/T-Y/W-D/T-S/T-A/S

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:110-119, or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain comprising an LC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 110-119.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 57-66, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76, or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and anHC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a light chainvariable domain comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions, an LC-CDR2 comprising the amino acid sequence of any oneof SEQ ID NOs: 100-109, or a variant thereof comprising up to about 3(such as about any of 1, 2, or 3) amino acid substitutions, and anLC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:110-119, or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 57-66, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76, or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and anHC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; and ii) a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 90-99, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109, or a variant thereofcomprising up to about 3 (such as about any of 1, 2, or 3) amino acidsubstitutions, and an LC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 110-119.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain sequence comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66; an HC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 67-76; andan HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences;and ii) a light chain variable domain sequence comprising an LC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 90-99; anLC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:100-109; and an LC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 110-119; or a variant thereof comprising up to about 5 (suchas about any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDRsequences.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain sequence comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66; an HC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 67-76; andan HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acidsubstitutions are in HC-CDR1 or HC-CDR2; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, wherein the amino acid substitutions are inHC-CDR1 or HC-CDR2.

In some embodiments, the anti-AMC antibody moiety comprises i) a heavychain variable domain sequence comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66; an HC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 67-76; andan HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; and ii) a light chain variable domain sequence comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99; an LC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 100-109; and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119. The sequences of the HC-CDRs notedherein are provided in Table 3 below and the LC-CDRs noted herein areprovided in Table 4 below.

TABLE 3  SEQ ID NO: 57 GYTFTSYG SEQ ID NO: 67 ISAYNGNT SEQ ID NO: 77ARYQDVVWYLGQFDQ 17 HC-CDR1 17 HC-CDR2 17 HC-CDR3 SEQ ID NO: 58 VSSNSAAWNSEQ ID NO: 68 YRSKVVYN SEQ ID NO: 78 ARGSYYSGRYDA 33 HC-CDR1 33 HC-CDR233 HC-CDR3 SEQ ID NO: 59 GGTFSSYA SEQ ID NO: 69 IIPIFGTA SEQ ID NO: 79AREIRGYYYYYGMDV 44 HC-CDR1 44 HC-CDR2 44 HC-CDR3 SEQ ID NO: 60 GFTFDDYASEQ ID NO: 70 ISWNSGRI SEQ ID NO: 80 ARADDYGAPYYYYGMDV 48 HC-CDR148 HC-CDR2 48 HC-CDR3 SEQ ID NO: 61 GGSISSSNW SEQ ID NO: 71 IYHSGSTSEQ ID NO: 81 ATGYGGYFDY 50 HC-CDR1 50 HC-CDR2 50 HC-CDR3 SEQ ID NO: 62GYTFTSYG SEQ ID NO: 72 ISAYNGNT SEQ ID NO: 82 ARDSYYYYYGMDV 52 HC-CDR152 HC-CDR2 52 HC-CDR3 SEQ ID NO: 63 GYSFPNYW SEQ ID NO: 73 IDPGDSYTSEQ ID NO: 83 ARYYVSLVDI 61 HC-CDR1 61 HC-CDR2 61 HC-CDR3 SEQ ID NO: 64GFTFSNAW SEQ ID NO: 74 IRSKAYGGTT SEQ ID NO: 84 ARDGLYSSSVVYDSDY76 HC-CDR1 76 HC-CDR2 76 HC-CDR3 SEQ ID NO: 65 GFTFDDYA SEQ ID NO: 75ISWNSGSI SEQ ID NO: 85 AKDIHSGSYYGLLYYAMDV 79 HC-CDR1 79 HC-CDR279 HC-CDR3 SEQ ID NO: 66 GYTFTSYG SEQ ID NO: 76 ISAYNGNT SEQ ID NO:86ARFQDVVWYLGQFDQ 17-13 HC- 17-13 HC- 17-13 HC- CDR1 CDR2 CDR3

TABLE 4  SEQ ID NO: 90 GSDVGVYYY SEQ ID NO: 100 DVG SEQ ID NO: 110ASYTNRNSLGYV 17 LC-CDR1 17 LC-CDR2 17 LC-CDR3 SEQ ID NO: 91 SGSIASNYSEQ ID NO: 101 EDN SEQ ID NO: 111 QSYDSSTVV 33 LC-CDR1 33 LC-CDR233 LC-CDR3 SEQ ID NO: 92 NIGTKS SEQ ID NO: 102 YDT SEQ ID NO: 112QVWDSSSDHPV 44 LC-CDR1 44 LC-CDR2 44 LC-CDR3 SEQ ID NO: 93 SSNIGAGYDSEQ ID NO: 103 GNS SEQ ID NO: 113 QSYDSSLSGSV 48 LC-CDR1 48 LC-CDR248 LC-CDR3 SEQ ID NO: 94 NIGSKS SEQ ID NO: 104 YDS SEQ ID NO: 114QVWDSSSDHVV 50 LC-CDR1 50 LC-CDR2 50 LC-CDR3 SEQ ID NO: 95 TGAVTSGHYSEQ ID NO: 105 DAS SEQ ID NO: 115 LLSYSDALV 52 LC-CDR1 52 LC-CDR252 LC-CDR3 SEQ ID NO: 96 SSDVGGYNY SEQ ID NO: 106 DVN SEQ ID NO: 116SSYTTGSRAV 61 LC-CDR1 61 LC-CDR2 61 LC-CDR3 SEQ ID NO: 97 SSNIGNNYSEQ ID NO: 107 DNN SEQ ID NO: 117 GTWDGSLYTML 76 LC-CDR1 76 LC-CDR276 LC-CDR3 SEQ ID NO: 98 SSNIGAGYD SEQ ID NO: 108 GNS SEQ ID NO: 118QSYDSSLSGSGV 79 LC-CDR1 79 LC-CDR2 79 LC-CDR3 SEQ ID NO: 99 GSDVGVYYYSEQ ID NO: 109 DVD SEQ ID NO:119 ASYTNRNSLGYV 17-13 LC-CDR117-13 LC-CDR2 17-13 LC-CDR3

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence of any one ofSEQ ID NOs: 17-26, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity, and a light chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 27-36, or a variantthereof having at least about 95% (including for example at least any of96%, 97%, 98%, or 99%) sequence identity.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence of any one ofSEQ ID NOs: 17-26 and a light chain variable domain comprising the aminoacid sequence of any one of SEQ ID NOs: 27-36.

The heavy and light chain variable domains can be combined in variouspair-wise combinations to generate a number of anti-AMC antibodymoieties.

For example, in some embodiments, the anti-AMC antibody moiety comprisesa heavy chain variable domain comprising an HC-CDR1 comprising the aminoacid sequence of SEQ ID NO: 57, or a variant thereof comprising up toabout 5 (for example about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; an HC-CDR2 comprising the amino acid sequence of SEQ IDNO: 67, or a variant thereof comprising up to about 5 (for example aboutany of 1, 2, 3, 4, or 5) amino acid substitutions; and an HC-CDR3comprising the amino acid sequence of SEQ ID NO: 77, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions; and a light chain variable domain comprisingan LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 90, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; an LC-CDR2 comprising the aminoacid sequence of SEQ ID NO: 100, or a variant thereof comprising up toabout 3 (for example about any of 1, 2, or 3) amino acid substitutions;and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 110, ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 57, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 67, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 77, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 90, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 100, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 110, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 57, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 67, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 77; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 90, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 100, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 58, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 68, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 78, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 91, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 101, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 111, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 58, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 68, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 78, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 91, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 111, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 58, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 68, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 78; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 91, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 111.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 59, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 79, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 92, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 102, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 112, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 59, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 69, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 79, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 92, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 102, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 112, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 59, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 69, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 79; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 92, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 102, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 112.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 60, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 80, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 93, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 103, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 113, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 60, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 70, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 80, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 93, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 103, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 113, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 60, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 70, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 80; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 93, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 103, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 113.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 61, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 71, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 81, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 94, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 104, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 114, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 61, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 71, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 81, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 94, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 104, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 114, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 61, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 71, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 81; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 94, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 104, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 114.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 62, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 82, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 95, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 105, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 115, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 62, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 72, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 82, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 95, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 105, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 115, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 62, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 72, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 82; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 95, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 105, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 115.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 63, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 83, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 96, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 106, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 116, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 63, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 73, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 83, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 96, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 116, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 63, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 73, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 83; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 96, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 106, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 116.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 64, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 74, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 84, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 97, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 107, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 117, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 64, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 74, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 84, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 97, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 107, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 117, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 64, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 74, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 84; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 97, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 107, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 117.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 65, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 75, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 85, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 98, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 108, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 118, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 65, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 75, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 85, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 98, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 108, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 118, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 65, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 75, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 85; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 98, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 108, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 118.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 66, or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions; anHC-CDR2 comprising the amino acid sequence of SEQ ID NO: 76, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; and an HC-CDR3 comprising theamino acid sequence of SEQ ID NO: 86, or a variant thereof comprising upto about 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 99, or a variantthereof comprising up to about 5 (for example about any of 1, 2, 3, 4,or 5) amino acid substitutions; an LC-CDR2 comprising the amino acidsequence of SEQ ID NO: 109, or a variant thereof comprising up to about3 (for example about any of 1, 2, or 3) amino acid substitutions; and anLC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 66, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 76, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 86, or a variant thereof comprising up to about 5 (such asabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 99, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 109, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 119, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions in the LC-CDR sequences.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising an HC-CDR1 comprising the amino acidsequence of SEQ ID NO: 66, an HC-CDR2 comprising the amino acid sequenceof SEQ ID NO: 76, and an HC-CDR3 comprising the amino acid sequence ofSEQ ID NO: 86; and a light chain variable domain comprising an LC-CDR1comprising the amino acid sequence of SEQ ID NO: 99, an LC-CDR2comprising the amino acid sequence of SEQ ID NO: 109, and an LC-CDR3comprising the amino acid sequence of SEQ ID NO: 119.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 17, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 27, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 17 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 27.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 18, or a variant thereof having at least about 95% (includingfor example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 28, or a variant thereof having atleast about 95% (including for example at least about any of 96%, 97%,98%, or 99%) sequence identity. In some embodiments, the anti-AMCantibody moiety comprises a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO: 18 and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO: 28.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 19, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 29, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 29.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 20, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 30, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 20 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 30.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 21, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 31, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 21 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 31.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 22, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 32, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 22 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 32.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 23, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 33, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 23 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 33.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 24, or a variant thereof having at least about 95% (includingfor example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 34, or a variant thereof having atleast about 95% (including for example at least about any of 96%, 97%,98%, or 99%) sequence identity. In some embodiments, the anti-AMCantibody moiety comprises a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO: 24 and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO: 34.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 25, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 35, or a variant thereof having at least about 95%(including for example at least about any of 96%, 97%, 98%, or 99%)sequence identity. In some embodiments, the anti-AMC antibody moietycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 25 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 35.

In some embodiments, the anti-AMC antibody moiety comprises a heavychain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 26, or a variant thereof having at least about 95% (includingfor example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 36, or a variant thereof having atleast about 95% sequence identity. In some embodiments, the anti-AMCantibody moiety comprises a heavy chain variable domain comprising theamino acid sequence set forth in SEQ ID NO: 26 and a light chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO: 36.

In some embodiments, the anti-AMC antibody moiety competes for bindingto a target AFP/MHC class I complex with a second anti-AMC antibodymoiety according to any of the anti-AMC antibody moieties describedherein. In some embodiments, the anti-AMC antibody moiety binds to thesame, or substantially the same, epitope as the second anti-AMC antibodymoiety. In some embodiments, binding of the anti-AMC antibody moiety tothe target AFP/MHC class I complex inhibits binding of the secondanti-AMC antibody moiety to the target AFP/MHC class I complex by atleast about 70% (such as by at least about any of 75%, 80%, 85%, 90%,95%, 98% or 99%), or vice versa. In some embodiments, the anti-AMCantibody moiety and the second anti-AMC antibody moiety cross-competefor binding to the target AFP/MHC class I complex, i.e., each of theantibody moieties competes with the other for binding to the targetAFP/MHC class I complex.

Full-Length Anti-AMC Antibodies

The anti-AMC constructs in some embodiments are full-length antibodiescomprising an anti-AMC antibody moiety (also referred to herein as a“full-length anti-AMC antibody”). In some embodiments, the full-lengthantibody is a monoclonal antibody.

In some embodiments, the full-length anti-AMC antibody comprises an Fcsequence from an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM. Insome embodiments, the full-length anti-AMC antibody comprises an Fcsequence of IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In someembodiments, the full-length anti-AMC antibody comprises an Fc sequenceof a human immunoglobulin. In some embodiments, the full-length anti-AMCantibody comprises an Fc sequence of a mouse immunoglobulin. In someembodiments, the full-length anti-AMC antibody comprises an Fc sequencethat has been altered or otherwise changed so that it has enhancedantibody dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC) effector function.

Thus, for example, in some embodiments, there is provided a full-lengthanti-AMC antibody comprising a) an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) an Fc region. In some embodiments, the AFPpeptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC class Iprotein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01. In some embodiments, there is provided a full-lengthanti-AMC antibody comprising a) an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, and b) an Fc region. In some embodiments, the Fcregion comprises an IgG1 Fc sequence. In some embodiments, the Fc regioncomprises a human IgG1 Fc sequence. In some embodiments, the Fc regioncomprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W(SEQ ID NO: 87), or a variant thereof comprising up to about 3 (forexample about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO:88), or a variant thereof comprising up to about 3 (for example aboutany of 1, 2, or 3) amino acid substitutions, and an HC-CDR3 comprisingthe amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); or avariant thereof comprising up to about 3 (for example about any of 1, 2,or 3) amino acid substitutions; and ii) a light chain variable domaincomprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) an Fc region. Insome embodiments, the Fc region comprises an IgG1 Fc sequence. In someembodiments, the Fc region comprises a human IgG1 Fc sequence. In someembodiments, the Fc region comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W(SEQ ID NO: 87), an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and an HC-CDR3 comprising theamino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii)a light chain variable domain comprising an LC-CDR1 comprising the aminoacid sequence of S/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), andan LC-CDR3 comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S(SEQ ID NO: 121); wherein X can be any amino acid, and b) an Fc region.In some embodiments, the Fc region comprises an IgG1 Fc sequence. Insome embodiments, the Fc region comprises a human IgG1 Fc sequence. Insome embodiments, the Fc region comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, an HC-CDR2 comprising the amino acid sequenceof any one of SEQ ID NOs: 67-76, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86, or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; andii) a light chain variable domain comprising an LC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 90-99, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, an LC-CDR2 comprising the amino acid sequenceof any one of SEQ ID NOs: 100-109, or a variant thereof comprising up toabout 3 (such as about any of 1, 2, or 3) amino acid substitutions, andan LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:110-119, or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments,the Fc region comprises an IgG1 Fc sequence. In some embodiments, the Fcregion comprises a human IgG1 Fc sequence. In some embodiments, the Fcregion comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66; anHC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:67-76; and an HC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 77-86; or a variant thereof comprising up to about 5 (suchas about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and ii) a light chain variable domain sequence comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99; an LC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 100-109; and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119; or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutionsin the LC-CDR sequences; and b) an Fc region. In some embodiments, theFc region comprises an IgG1 Fc sequence. In some embodiments, the Fcregion comprises a human IgG1 Fc sequence. In some embodiments, the Fcregion comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66; anHC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:67-76; and an HC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 77-86; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; and b) an Fc region. In someembodiments, the Fc region comprises an IgG1 Fc sequence. In someembodiments, the Fc region comprises a human IgG1 Fc sequence. In someembodiments, the Fc region comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisinga heavy chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 17-26, or a variant thereof having at least about 95%(for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a light chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 27-36, or a variant thereof having atleast about 95% sequence identity; and b) an Fc region. In someembodiments, the Fc region comprises an IgG1 Fc sequence. In someembodiments, the Fc region comprises a human IgG1 Fc sequence. In someembodiments, the Fc region comprises a mouse IgG1 Fc sequence.

In some embodiments, there is provided a full-length anti-AMC antibodycomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisinga heavy chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 17-26 and a light chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 27-36; and b) an Fcregion. In some embodiments, the Fc region comprises an IgG1 Fcsequence. In some embodiments, the Fc region comprises a human IgG1 Fcsequence. In some embodiments, the Fc region comprises a mouse IgG1 Fcsequence.

In some embodiments, the full-length anti-AMC antibody binds to acomplex comprising an AFP peptide and an MHC class I protein with aK_(d) between about 0.1 pM to about 500 nM (such as about any of 0.1 pM,1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100 nM, or 500nM, including any ranges between these values). In some embodiments, thefull-length anti-AMC antibody binds to a complex comprising an AFPpeptide and an MHC class I protein with a K_(d) between about 1 pM toabout 250 pM (such as about any of 1, 10, 25, 50, 75, 100, 150, 200, or250 pM, including any ranges between these values).

Multi-Specific Anti-AMC Molecules

The anti-AMC constructs in some embodiments comprise a multi-specificanti-AMC molecule comprising an anti-AMC antibody moiety and a secondbinding moiety (such as a second antigen-binding moiety). In someembodiments, the multi-specific anti-AMC molecule comprises an anti-AMCantibody moiety and a second antigen-binding moiety.

Multi-specific molecules are molecules that have binding specificitiesfor at least two different antigens or epitopes (e.g., bispecificantibodies have binding specificities for two antigens or epitopes).Multi-specific molecules with more than two valencies and/orspecificities are also contemplated. For example, trispecific antibodiescan be prepared. Tutt et al. J. Immunol. 147: 60 (1991). It is to beappreciated that one of skill in the art could select appropriatefeatures of individual multi-specific molecules described herein tocombine with one another to form a multi-specific anti-AMC molecule ofthe invention.

Thus, for example, in some embodiments, there is provided amulti-specific (e.g., bispecific) anti-AMC molecule comprising a) ananti-AMC antibody moiety that specifically binds to a complex comprisingan AFP peptide and an MHC class I protein, and b) a second bindingmoiety (such as an antigen-binding moiety). In some embodiments, thesecond binding moiety specifically binds to a complex comprising adifferent AFP peptide bound to the MHC class I protein. In someembodiments, the second scFv specifically binds to a complex comprisingthe AFP peptide bound to a different MHC class I protein. In someembodiments, the second binding moiety specifically binds to a differentepitope on the complex comprising the AFP peptide bound to the MHC classI protein. In some embodiments, the second binding moiety specificallybinds to a different antigen. In some embodiments, the second bindingmoiety specifically binds to an antigen on the surface of a cell, suchas a cytotoxic cell. In some embodiments, the second binding moietyspecifically binds to an antigen on the surface of a lymphocyte, such asa T cell, an NK cell, a neutrophil, a monocyte, a macrophage, or adendritic cell. In some embodiments, the second binding moietyspecifically binds to an effector T cell, such as a cytotoxic T cell(also known as cytotoxic T lymphocyte (CTL) or T killer cell).

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I protein,and b) a second antigen-binding moiety that binds specifically to CD3.In some embodiments, the second antigen-binding moiety specificallybinds to CD3ε. In some embodiments, the second antigen-binding moietyspecifically binds to an agonistic epitope of CD3ε. The term “agonisticepitope”, as used herein, means (a) an epitope that, upon binding of themulti-specific molecule, optionally upon binding of severalmulti-specific molecules on the same cell, allows said multi-specificmolecules to activate TCR signaling and induce T cell activation, and/or(b) an epitope that is solely composed of amino acid residues of theepsilon chain of CD3 and is accessible for binding by the multi-specificmolecule, when presented in its natural context on T cells (i.e.surrounded by the TCR, the CD3γ chain, etc.), and/or (c) an epitopethat, upon binding of the multi-specific molecule, does not lead tostabilization of the spatial position of CD3ε relative to CD3γ.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I protein,and b) a second antigen-binding moiety that binds specifically to anantigen on the surface of an effector cell, including for example CD3γ,CD3δ, CD3ε, CD3ζ, CD28, CD16a, CD56, CD68, and GDS2D.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I protein,and b) a second antigen-binding moiety that binds specifically to acomponent of the complement system, such as C1q. C1q is a subunit of theC1 enzyme complex that activates the serum complement system.

In some embodiments, the second antigen-binding moiety specificallybinds to an Fc receptor. In some embodiments, the second antigen-bindingmoiety specifically binds to an Fcγ receptor (FcγR). The FcγR may be anFcγRIII present on the surface of natural killer (NK) cells or one ofFcγRI, FcγRIIA, FcγRIIBI, FcγRIIB2, and FcγRIIIB present on the surfaceof macrophages, monocytes, neutrophils and/or dendritic cells. In someembodiments, the second antigen-binding moiety is an Fc region orfunctional fragment thereof. A “functional fragment” as used in thiscontext refers to a fragment of an antibody Fc region that is stillcapable of binding to an FcR, in particular to an FcγR, with sufficientspecificity and affinity to allow an FcγR bearing effector cell, inparticular a macrophage, a monocyte, a neutrophil and/or a dendriticcell, to kill the target cell by cytotoxic lysis or phagocytosis. Afunctional Fc fragment is capable of competitively inhibiting thebinding of the original, full-length Fc portion to an FcR such as theactivating FcγRI. In some embodiments, a functional Fc fragment retainsat least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of its affinity to anactivating FcγR. In some embodiments, the Fc region or functionalfragment thereof is an enhanced Fc region or functional fragmentthereof. The term “enhanced Fc region”, as used herein, refers to an Fcregion that is modified to enhance Fc receptor-mediatedeffector-functions, in particular antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), andantibody-mediated phagocytosis. This can be achieved as known in theart, for example by altering the Fc region in a way that leads to anincreased affinity for an activating receptor (e.g. FcγRIIIA (CD16A)expressed on natural killer (NK) cells) and/or a decreased binding to aninhibitory receptor (e.g. FcγRIIB1/B2 (CD32B)). In yet otherembodiments, the second antigen-binding moiety is an antibody orantigen-binding fragment thereof that specifically binds to an FcR, inparticular to an FcγR, with sufficient specificity and affinity to allowan FcγR bearing effector cell, in particular a macrophage, a monocyte, aneutrophil and/or a dendritic cell, to kill the target cell by cytotoxiclysis or phagocytosis.

In some embodiments, the multi-specific anti-AMC molecule allows killingof AMC-presenting target cells and/or can effectively redirect CTLs tolyse AMC-presenting target cells. In some embodiments, themulti-specific (e.g., bispecific) anti-AMC molecule of the presentinvention shows an in vitro EC₅₀ ranging from 10 to 500 ng/ml, and isable to induce redirected lysis of about 50% of the target cells throughCTLs at a ratio of CTLs to target cells of from about 1:1 to about 50:1(such as from about 1:1 to about 15:1, or from about 2:1 to about 10:1).

In some embodiments, the multi-specific (e.g., bispecific) anti-AMCmolecule is capable of cross-linking a stimulated or unstimulated CTLand the target cell in such a way that the target cell is lysed. Thisoffers the advantage that no generation of target-specific T cell clonesor common antigen presentation by dendritic cells is required for themulti-specific anti-AMC molecule to exert its desired activity. In someembodiments, the multi-specific anti-AMC molecule of the presentinvention is capable of redirecting CTLs to lyse the target cells in theabsence of other activating signals. In some embodiments, the secondantigen-binding moiety of the multi-specific anti-AMC moleculespecifically binds to CD3 (e.g., specifically binds to CD3ε), andsignaling through CD28 and/or IL-2 is not required for redirecting CTLsto lyse the target cells.

Methods for measuring the preference of the multi-specific anti-AMCmolecule to simultaneously bind to two antigens (e.g., antigens on twodifferent cells) are within the normal capabilities of a person skilledin the art. For example, when the second binding moiety specificallybinds to CD3, the multi-specific anti-AMC molecule may be contacted witha mixture of CD3⁺/AFP⁻ cells and CD3⁻/AFP⁺ cells. The number ofmulti-specific anti-AMC molecule-positive single cells and the number ofcells cross-linked by multi-specific anti-AMC molecules may then beassessed by microscopy or fluorescence-activated cell sorting (FACS) asknown in the art.

For example, in some embodiments, there is provided a multi-specificanti-AMC molecule comprising a) an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) a second antigen-binding moiety. In someembodiments, the AFP peptide is AFP158 (SEQ ID NO: 4). In someembodiments, the MHC class I protein is HLA-A02. In some embodiments,the MHC class I protein is HLA-A*02:01. In some embodiments, the secondantigen-binding moiety specifically binds to a complex comprising adifferent AFP peptide bound to the MHC class I protein. In someembodiments, the second antigen-binding moiety specifically binds to acomplex comprising the AFP peptide bound to a different MHC class Iprotein. In some embodiments, the second antigen-binding moietyspecifically binds to a different epitope on the complex comprising theAFP peptide bound to the MHC class I protein. In some embodiments, thesecond antigen-binding moiety specifically binds to another antigen. Insome embodiments, the second antigen-binding moiety specifically bindsto an antigen on the surface of a cell, such as an AMC-presenting cell.In some embodiments, the second antigen-binding moiety specificallybinds to an antigen on the surface of a cell that does not express AFP.In some embodiments, the second antigen-binding moiety specificallybinds to an antigen on the surface of a cytotoxic cell. In someembodiments, the second antigen-binding moiety specifically binds to anantigen on the surface of a lymphocyte, such as a T cell, an NK cell, aneutrophil, a monocyte, a macrophage, or a dendritic cell. In someembodiments, the second antigen-binding moiety specifically binds to anantigen on the surface of an effector T cell, such as a cytotoxic Tcell. In some embodiments, the second antigen-binding moietyspecifically binds to an antigen on the surface of an effector cell,including for example CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD16a, CD56, CD68,and GDS2D. In some embodiments, the anti-AMC antibody moiety is human,humanized, or semi-synthetic. In some embodiments, the secondantigen-binding moiety is an antibody moiety. In some embodiments, thesecond antigen-binding moiety is a human, humanized, or semi-syntheticantibody moiety. In some embodiments, the multi-specific anti-AMCmolecule further comprises at least one (such as at least about any of2, 3, 4, 5, or more) additional antigen-binding moieties.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP158 peptide (SEQ ID NO: 4) andHLA-A*02:01, and b) a second antigen-binding moiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) a secondantigen-binding moiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, and b) a second antigen-bindingmoiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 67-76, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 77-86, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and b) a second antigen-binding moiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in theHC-CDR sequences; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the LC-CDR sequences; and b) a secondantigen-binding moiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety that specificallybinds to a complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; and b) a secondantigen-binding moiety.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety comprising a heavychain variable domain comprising the amino acid sequence of any one ofSEQ ID NOs: 17-26, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity,and a light chain variable domain comprising the amino acid sequence ofany one of SEQ ID NOs: 27-36, or a variant thereof having at least about95% sequence identity; and b) a second scFv.

In some embodiments, there is provided a multi-specific anti-AMCmolecule comprising a) an anti-AMC antibody moiety comprising a heavychain variable domain comprising the amino acid sequence of any one ofSEQ ID NOs: 17-26 and a light chain variable domain comprising the aminoacid sequence of any one of SEQ ID NOs: 27-36; and b) a secondantigen-binding moiety.

In some embodiments, the multi-specific anti-AMC molecule is, forexample, a diabody (Db), a single-chain diabody (scDb), a tandem scDb(Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb(CD-scDb), a di-diabody, a tandem scFv, a tandem di-scFv (e.g., abispecific T cell engager), a tandem tri-scFv, a tri(a)body, abispecific Fab2, a di-miniantibody, a tetrabody, an scFv-Fc-scFv fusion,a dual-affinity retargeting (DART) antibody, a dual variable domain(DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-Fc, an IgG-scFvfusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH) antibody(bispecific IgG prepared by the KiH technology), a DuoBody (bispecificIgG prepared by the Duobody technology), a heteromultimeric antibody, ora heteroconjugate antibody. In some embodiments, the multi-specificanti-AMC molecule is a tandem scFv (e.g., a tandem di-scFv, such as abispecific T cell engager).

Tandem scFv

The multi-specific anti-AMC molecule in some embodiments is a tandemscFv comprising a first scFv comprising an anti-AMC antibody moiety anda second scFv (also referred to herein as a “tandem scFv multi-specificanti-AMC antibody”). In some embodiments, the tandem scFv multi-specificanti-AMC antibody further comprises at least one (such as at least aboutany of 2, 3, 4, 5, or more) additional scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) a second scFv. In some embodiments, the AFPpeptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC class Iprotein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01. In some embodiments, the second scFv specifically binds toa complex comprising a different AFP peptide bound to the MHC class Iprotein. In some embodiments, the second scFv specifically binds to acomplex comprising the AFP peptide bound to a different MHC class Iprotein. In some embodiments, the second scFv specifically binds to adifferent epitope on the complex comprising the AFP peptide bound to theMHC class I protein. In some embodiments, the second scFv specificallybinds to another antigen. In some embodiments, the second scFvspecifically binds to an antigen on the surface of a cell, such as anAMC-presenting cell. In some embodiments, the second scFv specificallybinds to an antigen on the surface of a cell that does not express AFP.In some embodiments, the second scFv specifically binds to an antigen onthe surface of a cytotoxic cell. In some embodiments, the second scFvspecifically binds to an antigen on the surface of a lymphocyte, such asa T cell, an NK cell, a neutrophil, a monocyte, a macrophage, or adendritic cell. In some embodiments, the second scFv specifically bindsto an antigen on the surface of an effector T cell, such as a cytotoxicT cell. In some embodiments, the second scFv specifically binds to anantigen on the surface of an effector cell, including for example CD3γ,CD3δ, CD3ε, CD3ζ, CD28, CD16a, CD56, CD68, and GDS2D. In someembodiments, the first scFv is human, humanized, or semi-synthetic. Insome embodiments, the second scFv is human, humanized, orsemi-synthetic. In some embodiments, both the first scFv and the secondscFv are human, humanized, or semi-synthetic. In some embodiments, thetandem scFv multi-specific anti-AMC antibody further comprises at leastone (such as at least about any of 2, 3, 4, 5, or more) additional scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, and b) a second scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) a second scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, and b) a second scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingan HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and b) a second scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the HC-CDR sequences; and ii) a light chainvariable domain sequence comprising an LC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 110-119; ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions in the LC-CDR sequences; and b) asecond scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; and b) asecond scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFvcomprising a heavy chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 17-26, or a variant thereof having atleast about 95% (for example at least about any of 96%, 97%, 98%, or99%) sequence identity, and a light chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 27-36, or a variantthereof having at least about 95% sequence identity; and b) a secondscFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFvcomprising a heavy chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 17-26 and a light chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:27-36; and b) a second scFv.

In some embodiments, there is provided a tandem scFv multi-specific(e.g., bispecific) anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) a second scFv, wherein the tandem scFvmulti-specific anti-AMC antibody is a tandem di-scFv or a tandemtri-scFv. In some embodiments, the tandem scFv multi-specific anti-AMCantibody is a tandem di-scFv. In some embodiments, the tandem scFvmulti-specific anti-AMC antibody is a bispecific T-cell engager.

For example, in some embodiments, there is provided a tandem di-scFvbispecific anti-AMC antibody comprising a) a first scFv thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) a second scFv that specifically binds to anantigen on the surface of a T cell. In some embodiments, the AFP peptideis AFP158 (SEQ ID NO: 4). In some embodiments, the MHC class I proteinis HLA-A02. In some embodiments, the MHC class I protein is HLA-A*02:01.In some embodiments, the second scFv specifically binds to an antigen onthe surface of an effector T cell, such as a cytotoxic T cell. In someembodiments, the second scFv specifically binds to an antigen selected,for example, from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD28,OX40, GITR, CD137, CD27, CD40L, and HVEM. In some embodiments, thesecond scFv specifically binds to an agonistic epitope on an antigen onthe surface of a T cell, wherein the binding of the second scFv to theantigen enhances T cell activation. In some embodiments, the first scFvis human, humanized, or semi-synthetic. In some embodiments, the secondscFv is human, humanized, or semi-synthetic. In some embodiments, boththe first scFv and the second scFv are human, humanized, orsemi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP158 peptide (SEQ ID NO: 4) and HLA-A*02:01,and b) a second scFv that specifically binds to an antigen on thesurface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) a second scFvthat specifically binds to an antigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, and b) a second scFv thatspecifically binds to an antigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 67-76, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 77-86, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and b) a second scFv that specifically binds to anantigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in theHC-CDR sequences; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the LC-CDR sequences, and b) a second scFv thatspecifically binds to an antigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; and b) a second scFv thatspecifically binds to an antigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv comprising a heavy chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 17-26, or a variant thereof having at least about 95% (for exampleat least about any of 96%, 97%, 98%, or 99%) sequence identity, and alight chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 27-36, or a variant thereof having at least about 95%(for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and b) a second scFv that specifically binds to an antigen onthe surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv comprising a heavy chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 17-26 and a light chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 27-36, and b) a second scFv thatspecifically binds to an antigen on the surface of a T cell.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I protein, and b) asecond scFv that specifically binds to CD3ε. In some embodiments, theAFP peptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC classI protein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01. In some embodiments, the first scFv is fused to the secondscFv through linkage with a peptide linker. In some embodiments, thepeptide linker is between about 5 to about 20 (such as about any of 5,10, 15, or 20, including any ranges between these values) amino acids inlength. In some embodiments, the peptide linker comprises (and in someembodiments consists of) the amino acid sequence GGGGS. In someembodiments, the first scFv is human, humanized, or semi-synthetic. Insome embodiments, the second scFv is human, humanized, orsemi-synthetic. In some embodiments, both the first scFv and the secondscFv are human, humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP158 peptide (SEQ ID NO: 4) and HLA-A*02:01,and b) a second scFv that specifically binds to CD3ε. In someembodiments, the first scFv is fused to the second scFv through linkagewith a peptide linker. In some embodiments, the peptide linker isbetween about 5 to about 20 (such as about any of 5, 10, 15, or 20,including any ranges between these values) amino acids in length. Insome embodiments, the peptide linker comprises (and in some embodimentsconsists of) the amino acid sequence GGGGS. In some embodiments, thefirst scFv is human, humanized, or semi-synthetic. In some embodiments,the second scFv is human, humanized, or semi-synthetic. In someembodiments, both the first scFv and the second scFv are human,humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) a second scFvthat specifically binds to CD3ε. In some embodiments, the first scFv isfused to the second scFv through linkage with a peptide linker. In someembodiments, the peptide linker is between about 5 to about 20 (such asabout any of 5, 10, 15, or 20, including any ranges between thesevalues) amino acids in length. In some embodiments, the peptide linkercomprises (and in some embodiments consists of) the amino acid sequenceGGGGS. In some embodiments, the first scFv is human, humanized, orsemi-synthetic. In some embodiments, the second scFv is human,humanized, or semi-synthetic. In some embodiments, both the first scFvand the second scFv are human, humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, and b) a second scFv thatspecifically binds to CD3ε. In some embodiments, the first scFv is fusedto the second scFv through linkage with a peptide linker. In someembodiments, the peptide linker is between about 5 to about 20 (such asabout any of 5, 10, 15, or 20, including any ranges between thesevalues) amino acids in length. In some embodiments, the peptide linkercomprises (and in some embodiments consists of) the amino acid sequenceGGGGS. In some embodiments, the first scFv is human, humanized, orsemi-synthetic. In some embodiments, the second scFv is human,humanized, or semi-synthetic. In some embodiments, both the first scFvand the second scFv are human, humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66, or avariant thereof comprising up to about 5 (such as about any of 1, 2, 3,4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 67-76, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 77-86, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions, and b) a second scFv that specifically binds to CD3ε. Insome embodiments, the first scFv is fused to the second scFv throughlinkage with a peptide linker. In some embodiments, the peptide linkeris between about 5 to about 20 (such as about any of 5, 10, 15, or 20,including any ranges between these values) amino acids in length. Insome embodiments, the peptide linker comprises (and in some embodimentsconsists of) the amino acid sequence GGGGS. In some embodiments, thefirst scFv is human, humanized, or semi-synthetic. In some embodiments,the second scFv is human, humanized, or semi-synthetic. In someembodiments, both the first scFv and the second scFv are human,humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in theHC-CDR sequences; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the LC-CDR sequences, and b) a second scFv thatspecifically binds to CD3ε. In some embodiments, the first scFv is fusedto the second scFv through linkage with a peptide linker. In someembodiments, the peptide linker is between about 5 to about 20 (such asabout any of 5, 10, 15, or 20, including any ranges between thesevalues) amino acids in length. In some embodiments, the peptide linkercomprises (and in some embodiments consists of) the amino acid sequenceGGGGS. In some embodiments, the first scFv is human, humanized, orsemi-synthetic. In some embodiments, the second scFv is human,humanized, or semi-synthetic. In some embodiments, both the first scFvand the second scFv are human, humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv that specifically binds toa complex comprising an AFP peptide and an MHC class I proteincomprising i) a heavy chain variable domain sequence comprising anHC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66; an HC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 67-76; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; and b) a second scFv thatspecifically binds to CD3ε. In some embodiments, the first scFv is fusedto the second scFv through linkage with a peptide linker. In someembodiments, the peptide linker is between about 5 to about 20 (such asabout any of 5, 10, 15, or 20, including any ranges between thesevalues) amino acids in length. In some embodiments, the peptide linkercomprises (and in some embodiments consists of) the amino acid sequenceGGGGS. In some embodiments, the first scFv is human, humanized, orsemi-synthetic. In some embodiments, the second scFv is human,humanized, or semi-synthetic. In some embodiments, both the first scFvand the second scFv are human, humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv comprising a heavy chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 17-26, or a variant thereof having at least about 95% (for exampleat least about any of 96%, 97%, 98%, or 99%) sequence identity, and alight chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 27-36, or a variant thereof having at least about 95%(for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and b) a second scFv that specifically binds to CD3ε. In someembodiments, the first scFv is fused to the second scFv through linkagewith a peptide linker. In some embodiments, the peptide linker isbetween about 5 to about 20 (such as about any of 5, 10, 15, or 20,including any ranges between these values) amino acids in length. Insome embodiments, the peptide linker comprises (and in some embodimentsconsists of) the amino acid sequence GGGGS. In some embodiments, thefirst scFv is human, humanized, or semi-synthetic. In some embodiments,the second scFv is human, humanized, or semi-synthetic. In someembodiments, both the first scFv and the second scFv are human,humanized, or semi-synthetic.

In some embodiments, there is provided a tandem di-scFv bispecificanti-AMC antibody comprising a) a first scFv comprising a heavy chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 17-26 and a light chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 27-36, and b) a second scFv thatspecifically binds to CD3ε. In some embodiments, the first scFv is fusedto the second scFv through linkage with a peptide linker. In someembodiments, the peptide linker is between about 5 to about 20 (such asabout any of 5, 10, 15, or 20, including any ranges between thesevalues) amino acids in length. In some embodiments, the peptide linkercomprises (and in some embodiments consists of) the amino acid sequenceGGGGS. In some embodiments, the first scFv is human, humanized, orsemi-synthetic. In some embodiments, the second scFv is human,humanized, or semi-synthetic. In some embodiments, both the first scFvand the second scFv are human, humanized, or semi-synthetic.

In some embodiments, the tandem di-scFv bispecific anti-AMC antibodybinds to a complex comprising an AFP peptide and an MHC class I proteinwith a K_(d) between about 0.1 pM to about 500 nM (such as about any of0.1 pM, 1.0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM, 50 nM, 100nM, or 500 nM, including any ranges between these values). In someembodiments, the tandem di-scFv bispecific anti-AMC antibody binds to acomplex comprising an AFP peptide and an MHC class I protein with aK_(d) between about 1 nM to about 500 nM (such as about any of 1, 10,25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM, includingany ranges between these values).

Chimeric Antigen Receptor (CAR) and CAR Effector Cells

The anti-AMC construct in some embodiments is a chimeric antigenreceptor (CAR) comprising an anti-AMC antibody moiety (also referred toherein as an “anti-AMC CAR”). Also provided is a CAR effector cell(e.g., T cell) comprising a CAR comprising an anti-AMC antibody moiety(also referred to herein as an “anti-AMC CAR effector cell”, e.g.,“anti-AMC CAR T cell”).

The anti-AMC CAR comprises a) an extracellular domain comprising ananti-AMC antibody moiety that specifically binds to a complex comprisingan AFP peptide and an MHC class I protein and b) an intracellularsignaling domain. A transmembrane domain may be present between theextracellular domain and the intracellular domain.

Between the extracellular domain and the transmembrane domain of theanti-AMC CAR, or between the intracellular domain and the transmembranedomain of the anti-AMC CAR, there may be a spacer domain. The spacerdomain can be any oligo- or polypeptide that functions to link thetransmembrane domain to the extracellular domain or the intracellulardomain in the polypeptide chain. A spacer domain may comprise up toabout 300 amino acids, including for example about 10 to about 100, orabout 25 to about 50 amino acids.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the α, β, δ, or γ chain of theT-cell receptor, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In someembodiments, the transmembrane domain may be synthetic, in which case itmay comprise predominantly hydrophobic residues such as leucine andvaline. In some embodiments, a triplet of phenylalanine, tryptophan andvaline may be found at each end of a synthetic transmembrane domain. Insome embodiments, a short oligo- or polypeptide linker, having a lengthof, for example, between about 2 and about 10 (such as about any of 2,3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length may form the linkagebetween the transmembrane domain and the intracellular signaling domainof the anti-AMC CAR. In some embodiments, the linker is a glycine-serinedoublet.

In some embodiments, the transmembrane domain that naturally isassociated with one of the sequences in the intracellular domain of theanti-AMC CAR is used (e.g., if an anti-AMC CAR intracellular domaincomprises a CD28 co-stimulatory sequence, the transmembrane domain ofthe anti-AMC CAR is derived from the CD28 transmembrane domain). In someembodiments, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The intracellular signaling domain of the anti-AMC CAR is responsiblefor activation of at least one of the normal effector functions of theimmune cell in which the anti-AMC CAR has been placed in. Effectorfunction of a T cell, for example, may be cytolytic activity or helperactivity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term “intracellular signaling sequence” is thusmeant to include any truncated portion of the intracellular signalingdomain sufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the anti-AMC CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (co-stimulatory signaling sequences).

Primary signaling sequences regulate primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primarysignaling sequences that act in a stimulatory manner may containsignaling motifs which are known as immunoreceptor tyrosine-basedactivation motifs or ITAMs. The anti-AMC CAR constructs in someembodiments comprise one or more ITAMs.

Examples of ITAM containing primary signaling sequences that are ofparticular use in the invention include those derived from TCRζ, FcRγ,FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the anti-AMC CAR comprises a primary signalingsequence derived from CD3ζ. For example, the intracellular signalingdomain of the CAR can comprise the CD3ζ intracellular signaling sequenceby itself or combined with any other desired intracellular signalingsequence(s) useful in the context of the anti-AMC CAR of the invention.For example, the intracellular domain of the anti-AMC CAR can comprise aCD3ζ intracellular signaling sequence and a costimulatory signalingsequence. The costimulatory signaling sequence can be a portion of theintracellular domain of a costimulatory molecule including, for example,CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, and the like.

In some embodiments, the intracellular signaling domain of the anti-AMCCAR comprises the intracellular signaling sequence of CD3ζ and theintracellular signaling sequence of CD28. In some embodiments, theintracellular signaling domain of the anti-AMC CAR comprises theintracellular signaling sequence of CD3ζ and the intracellular signalingsequence of 4-1BB. In some embodiments, the intracellular signalingdomain of the anti-AMC CAR comprises the intracellular signalingsequence of CD3ζ and the intracellular signaling sequences of CD28 and4-1BB.

Thus, for example, in some embodiments, there is provided an anti-AMCCAR comprising a) an extracellular domain comprising an anti-AMCantibody moiety that specifically binds to a complex comprising an AFPpeptide and an MHC class I protein, b) a transmembrane domain, and c) anintracellular signaling domain. In some embodiments, the AFP peptide isAFP158 (SEQ ID NO: 4). In some embodiments, the MHC class I protein isHLA-A02. In some embodiments, the MHC class I protein is HLA-A*02:01. Insome embodiments, the intracellular signaling domain is capable ofactivating an immune cell. In some embodiments, the intracellularsignaling domain comprises a primary signaling sequence and aco-stimulatory signaling sequence. In some embodiments, the primarysignaling sequence comprises a CD3ζ intracellular signaling sequence. Insome embodiments, the co-stimulatory signaling sequence comprises a CD28intracellular signaling sequence. In some embodiments, the intracellulardomain comprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, b) a transmembrane domain, and c) an intracellularsignaling domain. In some embodiments, the intracellular signalingdomain is capable of activating an immune cell. In some embodiments, theintracellular signaling domain comprises a primary signaling sequenceand a co-stimulatory signaling sequence. In some embodiments, theprimary signaling sequence comprises a CD3ζ intracellular signalingsequence. In some embodiments, the co-stimulatory signaling sequencecomprises a CD28 intracellular signaling sequence. In some embodiments,the intracellular domain comprises a CD3ζ intracellular signalingsequence and a CD28 intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, b) a transmembranedomain, and c) an intracellular signaling domain. In some embodiments,the intracellular signaling domain is capable of activating an immunecell. In some embodiments, the intracellular signaling domain comprisesa primary signaling sequence and a co-stimulatory signaling sequence. Insome embodiments, the primary signaling sequence comprises a CD3ζintracellular signaling sequence. In some embodiments, theco-stimulatory signaling sequence comprises a CD28 intracellularsignaling sequence. In some embodiments, the intracellular domaincomprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); b) an intracellular signaling domain. In some embodiments, theintracellular signaling domain is capable of activating an immune cell.In some embodiments, the intracellular signaling domain comprises aprimary signaling sequence and a co-stimulatory signaling sequence. Insome embodiments, the primary signaling sequence comprises a CD3ζintracellular signaling sequence. In some embodiments, theco-stimulatory signaling sequence comprises a CD28 intracellularsignaling sequence. In some embodiments, the intracellular domaincomprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingan HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; b) a transmembrane domain, and c) an intracellularsignaling domain. In some embodiments, the intracellular signalingdomain is capable of activating an immune cell. In some embodiments, theintracellular signaling domain comprises a primary signaling sequenceand a co-stimulatory signaling sequence. In some embodiments, theprimary signaling sequence comprises a CD3ζ intracellular signalingsequence. In some embodiments, the co-stimulatory signaling sequencecomprises a CD28 intracellular signaling sequence. In some embodiments,the intracellular domain comprises a CD3ζ intracellular signalingsequence and a CD28 intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the HC-CDR sequences; and ii) a light chainvariable domain sequence comprising an LC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 110-119; ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions in the LC-CDR sequences; b) atransmembrane domain, and c) an intracellular signaling domain. In someembodiments, the intracellular signaling domain is capable of activatingan immune cell. In some embodiments, the intracellular signaling domaincomprises a primary signaling sequence and a co-stimulatory signalingsequence. In some embodiments, the primary signaling sequence comprisesa CD3ζ intracellular signaling sequence. In some embodiments, theco-stimulatory signaling sequence comprises a CD28 intracellularsignaling sequence. In some embodiments, the intracellular domaincomprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; b) anintracellular signaling domain. In some embodiments, the intracellularsignaling domain is capable of activating an immune cell. In someembodiments, the intracellular signaling domain comprises a primarysignaling sequence and a co-stimulatory signaling sequence. In someembodiments, the primary signaling sequence comprises a CD3ζintracellular signaling sequence. In some embodiments, theco-stimulatory signaling sequence comprises a CD28 intracellularsignaling sequence. In some embodiments, the intracellular domaincomprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26, or a variantthereof having at least about 95% (for example at least about any of96%, 97%, 98%, or 99%) sequence identity, and a light chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:27-36, or a variant thereof having at least about 95% sequence identity;b) a transmembrane domain, and c) an intracellular signaling domain. Insome embodiments, the intracellular signaling domain is capable ofactivating an immune cell. In some embodiments, the intracellularsignaling domain comprises a primary signaling sequence and aco-stimulatory signaling sequence. In some embodiments, the primarysignaling sequence comprises a CD3ζ intracellular signaling sequence. Insome embodiments, the co-stimulatory signaling sequence comprises a CD28intracellular signaling sequence. In some embodiments, the intracellulardomain comprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26 and a light chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 27-36; b) an intracellular signaling domain. In some embodiments,the intracellular signaling domain is capable of activating an immunecell. In some embodiments, the intracellular signaling domain comprisesa primary signaling sequence and a co-stimulatory signaling sequence. Insome embodiments, the primary signaling sequence comprises a CD3ζintracellular signaling sequence. In some embodiments, theco-stimulatory signaling sequence comprises a CD28 intracellularsignaling sequence. In some embodiments, the intracellular domaincomprises a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence. In some embodiments, the AFPpeptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC class Iprotein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, b) a transmembranedomain, and c) an intracellular signaling domain comprising a CD3ζintracellular signaling sequence and a CD28 intracellular signalingsequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, b) a transmembrane domain, and c)an intracellular signaling domain comprising a CD3ζ intracellularsignaling sequence and a CD28 intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingan HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the HC-CDR sequences; and ii) a light chainvariable domain sequence comprising an LC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 110-119; ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions in the LC-CDR sequences; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 17-26, or a variantthereof having at least about 95% (for example at least about any of96%, 97%, 98%, or 99%) sequence identity, and a light chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:27-36, or a variant thereof having at least about 95% sequence identity;b) a transmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

In some embodiments, there is provided an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26 and a light chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 27-36; b) a transmembrane domain, and c) an intracellular signalingdomain comprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence.

Also provided herein are effector cells (such as lymphocytes, e.g., Tcells) expressing an anti-AMC CAR.

Also provided is a method of producing an effector cell expressing ananti-AMC CAR, the method comprising introducing a vector comprising anucleic acid encoding the anti-AMC CAR into the effector cell. In someembodiments, introducing the vector into the effector cell comprisestransducing the effector cell with the vector. In some embodiments,introducing the vector into the effector cell comprises transfecting theeffector cell with the vector. Transduction or transfection of thevector into the effector cell can be carried about using any methodknown in the art.

Immunoconjugates

The anti-AMC constructs in some embodiments comprise an immunoconjugatecomprising an anti-AMC antibody moiety attached to an effector molecule(also referred to herein as an “anti-AMC immunoconjugate”). In someembodiments the effector molecule is a therapeutic agent, such as acancer therapeutic agent, which is either cytotoxic, cytostatic orotherwise provides some therapeutic benefit. In some embodiments, theeffector molecule is a label, which can generate a detectable signal,either directly or indirectly.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising an anti-AMC antibody moiety and a therapeutic agent (alsoreferred to herein as an “antibody-drug conjugate”, or “ADC”). In someembodiments, the therapeutic agent is a toxin that is either cytotoxic,cytostatic or otherwise prevents or reduces the ability of the targetcells to divide. The use of ADCs for the local delivery of cytotoxic orcytostatic agents, i.e., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos, Anticancer Research19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev.26:151-172 (1997); U.S. Pat. No. 4,975,278) allows targeted delivery ofthe drug moiety to target cells, and intracellular accumulation therein,where systemic administration of these unconjugated therapeutic agentsmay result in unacceptable levels of toxicity to normal cells as well asthe target cells sought to be eliminated (Baldwin et al., Lancet (Mar.15, 1986):603-605 (1986); Thorpe, (1985) “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84:Biological And Clinical Applications, A. Pinchera et al. (eds.), pp.475-506). Maximal efficacy with minimal toxicity is sought thereby.Importantly, since most normal cells do not present the AMC on theirsurface, they cannot bind the anti-AMC immunoconjugate, and areprotected from the killing effect of the toxin or other therapeuticagents.

Therapeutic agents used in anti-AMC immunoconjugates include, forexample, daunomycin, doxorubicin, methotrexate, and vindesine (Rowlandet al., Cancer Immunol. Immunother. 21:183-187 (1986)). Toxins used inanti-AMC immunoconjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581(2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028(2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)),maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). Thetoxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

Enzymatically active toxins and fragments thereof that can be usedinclude, for example, diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993.

Anti-AMC immunoconjugates of an anti-AMC antibody moiety and one or moresmall molecule toxins, such as a calicheamicin, maytansinoids,dolastatins, aurostatins, a trichothecene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a therapeutic agent that has an intracellular activity. Insome embodiments, the anti-AMC immunoconjugate is internalized and thetherapeutic agent is a cytotoxin that blocks the protein synthesis ofthe cell, therein leading to cell death. In some embodiments, thetherapeutic agent is a cytotoxin comprising a polypeptide havingribosome-inactivating activity including, for example, gelonin,bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin,restrictocin, Pseudomonas exotoxin A and variants thereof. In someembodiments, where the therapeutic agent is a cytotoxin comprising apolypeptide having a ribosome-inactivating activity, the anti-AMCimmunoconjugate must be internalized upon binding to the target cell inorder for the protein to be cytotoxic to the cells.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a therapeutic agent that acts to disrupt DNA. In someembodiments, the therapeutic agent that acts to disrupt DNA is, forexample, selected from the group consisting of enediyne (e.g.,calicheamicin and esperamicin) and non-enediyne small molecule agents(e.g., bleomycin, methidiumpropyl-EDTA-Fe(II)). Other cancer therapeuticagents useful in accordance with the present application include,without limitation, daunorubicin, doxorubicin, distamycin A, cisplatin,mitomycin C, ecteinascidins, duocarmycin/CC-1065, andbleomycin/pepleomycin.

The present invention further contemplates an anti-AMC immunoconjugateformed between the anti-AMC antibody moiety and a compound withnucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such asa deoxyribonuclease; DNase).

In some embodiments, the anti-AMC immunoconjugate comprises an agentthat acts to disrupt tubulin. Such agents may include, for example,rhizoxin/maytansine, paclitaxel, vincristine and vinblastine,colchicine, auristatin dolastatin 10 MMAE, and peloruside A.

In some embodiments, the anti-AMC immunoconjugate comprises analkylating agent including, for example, Asaley NSC 167780, AZQ NSC182986, BCNU NSC 409962, Busulfan NSC 750, carboxyphthalatoplatinum NSC271674, CBDCA NSC 241240, CCNU NSC 79037, CHIP NSC 256927, chlorambucilNSC 3088, chlorozotocin NSC 178248, cis-platinum NSC 119875, clomesoneNSC 338947, cyanomorpholinodoxorubicin NSC 357704, cyclodisone NSC348948, dianhydrogalactitol NSC 132313, fluorodopan NSC 73754, hepsulfamNSC 329680, hycanthone NSC 142982, melphalan NSC 8806, methyl CCNU NSC95441, mitomycin C NSC 26980, mitozolamide NSC 353451, nitrogen mustardNSC 762, PCNU NSC 95466, piperazine NSC 344007, piperazinedione NSC135758, pipobroman NSC 25154, porfiromycin NSC 56410, spirohydantoinmustard NSC 172112, teroxirone NSC 296934, tetraplatin NSC 363812,thio-tepa NSC 6396, triethylenemelamine NSC 9706, uracil nitrogenmustard NSC 34462, and Yoshi-864 NSC 102627.

In some embodiments, the cancer therapeutic agent portion of theanti-AMC immunoconjugate of the present application may comprise anantimitotic agent including, without limitation, allocolchicine NSC406042, Halichondrin B NSC 609395, colchicine NSC 757, colchicinederivative NSC 33410, dolastatin 10 NSC 376128 (NG-auristatin derived),maytansine NSC 153858, rhizoxin NSC 332598, taxol NSC 125973, taxolderivative NSC 608832, thiocolchicine NSC 361792, trityl cysteine NSC83265, vinblastine sulfate NSC 49842, and vincristine sulfate NSC 67574.

In some embodiments, the anti-AMC immunoconjugate comprises atopoisomerase I inhibitor including, without limitation, camptothecinNSC 94600, camptothecin, Na salt NSC 100880, aminocamptothecin NSC603071, camptothecin derivative NSC 95382, camptothecin derivative NSC107124, camptothecin derivative NSC 643833, camptothecin derivative NSC629971, camptothecin derivative NSC 295500, camptothecin derivative NSC249910, camptothecin derivative NSC 606985, camptothecin derivative NSC374028, camptothecin derivative NSC 176323, camptothecin derivative NSC295501, camptothecin derivative NSC 606172, camptothecin derivative NSC606173, camptothecin derivative NSC 610458, camptothecin derivative NSC618939, camptothecin derivative NSC 610457, camptothecin derivative NSC610459, camptothecin derivative NSC 606499, camptothecin derivative NSC610456, camptothecin derivative NSC 364830, camptothecin derivative NSC606497, and morpholinodoxorubicin NSC 354646.

In some embodiments, the anti-AMC immunoconjugate comprises atopoisomerase II inhibitor including, without limitation, doxorubicinNSC 123127, amonafide NSC 308847, m-AMSA NSC 249992, anthrapyrazolederivative NSC 355644, pyrazoloacridine NSC 366140, bisantrene HCL NSC337766, daunorubicin NSC 82151, deoxydoxorubicin NSC 267469,mitoxantrone NSC 301739, menogaril NSC 269148, N,N-dibenzyl daunomycinNSC 268242, oxanthrazole NSC 349174, rubidazone NSC 164011, VM-26 NSC122819, and VP-16 NSC 141540.

In some embodiments, the anti-AMC immunoconjugate comprises an RNA orDNA antimetabolite including, without limitation, L-alanosine NSC153353, 5-azacytidine NSC 102816, 5-fluorouracil NSC 19893, acivicin NSC163501, aminopterin derivative NSC 132483, aminopterin derivative NSC184692, aminopterin derivative NSC 134033, an antifol NSC 633713, anantifol NSC 623017, Baker's soluble antifol NSC 139105, dichlorallyllawsone NSC 126771, brequinar NSC 368390, ftorafur (pro-drug) NSC148958, 5,6-dihydro-5-azacytidine NSC 264880, methotrexate NSC 740,methotrexate derivative NSC 174121, N-(phosphonoacetyl)-L-aspartate(PALA) NSC 224131, pyrazofurin NSC 143095, trimetrexate NSC 352122, 3-HPNSC 95678, 2′-deoxy-5-fluorouridine NSC 27640, 5-HP NSC 107392, α-TGDRNSC 71851, aphidicolin glycinate NSC 303812, ara-C NSC 63878,5-aza-2′-deoxycytidine NSC 127716, β-TGDR NSC 71261, cyclocytidine NSC145668, guanazole NSC 1895, hydroxyurea NSC 32065, inosineglycodialdehyde NSC 118994, macbecin Il NSC 330500, pyrazoloimidazoleNSC 51143, thioguanine NSC 752, and thiopurine NSC 755.

In some embodiments, the anti-AMC immunoconjugate comprises a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include ²¹¹At,¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ²¹²Pb and radioactiveisotopes of Lu.

In some embodiments, the anti-AMC antibody moiety can be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

In some embodiments, an anti-AMC immunoconjugate may comprise ananti-AMC antibody moiety conjugated to a prodrug-activating enzyme. Insome such embodiments, a prodrug-activating enzyme converts a prodrug(e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an activedrug, such as an anti-cancer drug. Such anti-AMC immunoconjugates areuseful, in some embodiments, in antibody-dependent enzyme-mediatedprodrug therapy (“ADEPT”). Enzymes that may be conjugated to an antibodyinclude, but are not limited to, alkaline phosphatases, which are usefulfor converting phosphate-containing prodrugs into free drugs;arylsulfatases, which are useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase, which is useful forconverting non-toxic 5-fluorocytosine into the anti-cancer drug,5-fluorouracil; proteases, such as serratia protease, thermolysin,subtilisin, carboxypeptidases and cathepsins (such as cathepsins B andL), which are useful for converting peptide-containing prodrugs intofree drugs; D-alanylcarboxypeptidases, which are useful for convertingprodrugs that contain D-amino acid substituents; carbohydrate-cleavingenzymes such as β-galactosidase and neuraminidase, which are useful forconverting glycosylated prodrugs into free drugs; β-lactamase, which isuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. In some embodiments, enzymes may be covalently bound toantibody moieties by recombinant DNA techniques well known in the art.See, e.g., Neuberger et al., Nature 312:604-608 (1984).

In some embodiments, the therapeutic portion of the anti-AMCimmunoconjugates may be a nucleic acid. Nucleic acids that may be usedinclude, but are not limited to, anti-sense RNA, genes or otherpolynucleotides, including nucleic acid analogs such as thioguanine andthiopurine.

The present application further provides anti-AMC immunoconjugatescomprising an anti-AMC antibody moiety attached to an effector molecule,wherein the effector molecule is a label, which can generate adetectable signal, indirectly or directly. These anti-AMCimmunoconjugates can be used for research or diagnostic applications,such as for the in vivo detection of cancer. The label is preferablycapable of producing, either directly or indirectly, a detectablesignal. For example, the label may be radio-opaque or a radioisotope,such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore)or chemiluminescent (chromophore) compound, such as fluoresceinisothiocyanate, rhodamine or luciferin; an enzyme, such as alkalinephosphatase, β-galactosidase or horseradish peroxidase; an imagingagent; or a metal ion. In some embodiments, the label is a radioactiveatom for scintigraphic studies, for example ⁹⁹Tc or ¹²³I, or a spinlabel for nuclear magnetic resonance (NMR) imaging (also known asmagnetic resonance imaging, MRI), such as zirconium-89, iodine-123,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron. Zirconium-89 may be complexed to variousmetal chelating agents and conjugated to antibodies, e.g., for PETimaging (WO 2011/056983).

In some embodiments, the anti-AMC immunoconjugate is detectableindirectly. For example, a secondary antibody that is specific for theanti-AMC immunoconjugate and contains a detectable label can be used todetect the anti-AMC immunoconjugate.

Thus, for example, in some embodiments, there is provided an anti-AMCimmunoconjugate comprising a) an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, and b) an effector molecule. In some embodiments, theAFP peptide is AFP158 (SEQ ID NO: 4). In some embodiments, the MHC classI protein is HLA-A02. In some embodiments, the MHC class I protein isHLA-A*02:01. In some embodiments, the effector molecule is covalentlyattached to the anti-AMC antibody moiety. In some embodiments, theeffector molecule is a therapeutic agent selected, for example, from thegroup consisting of a drug, a toxin, a radioisotope, a protein, apeptide, and a nucleic acid. In some embodiments, the effector molecularis a cancer therapeutic agent. In some embodiments, the cancertherapeutic agent is a chemotherapeutic. In some embodiments, the cancertherapeutic agent is a highly radioactive atom selected, for example,from the group consisting of ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ²¹²Bi, ³²P, and ²¹²Pb. In some embodiments, the effector moleculeis a label that can generate a detectable signal, either directly orindirectly. In some embodiments, the label is a radioisotope selected,for example, from the group consisting of ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I,and ¹³¹I. In some embodiments, the anti-AMC antibody moiety is an scFv.In some embodiments, the anti-AMC antibody moiety is human, humanized,or semi-synthetic.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP158 peptide (SEQ ID NO: 4) and HLA-A*02:01, andb) an effector molecule. In some embodiments, the effector molecule iscovalently attached to the anti-AMC antibody moiety. In someembodiments, the effector molecule is a therapeutic agent selected, forexample, from the group consisting of a drug, a toxin, a radioisotope, aprotein, a peptide, and a nucleic acid. In some embodiments, theeffector molecular is a cancer therapeutic agent. In some embodiments,the cancer therapeutic agent is a chemotherapeutic. In some embodiments,the cancer therapeutic agent is a highly radioactive atom selected, forexample, from the group consisting of ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, and ²¹²Pb. In some embodiments, the effectormolecule is a label that can generate a detectable signal, eitherdirectly or indirectly. In some embodiments, the label is a radioisotopeselected, for example, from the group consisting of ³H, ¹⁴C, ³²P, ³⁵S,¹²³I, ¹²⁵I, and ¹³¹I. In some embodiments, the anti-AMC antibody moietyis an scFv. In some embodiments, the anti-AMC antibody moiety is human,humanized, or semi-synthetic.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W(SEQ ID NO: 87), or a variant thereof comprising up to about 3 (forexample about any of 1, 2, or 3) amino acid substitutions, an HC-CDR2comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO:88), or a variant thereof comprising up to about 3 (for example aboutany of 1, 2, or 3) amino acid substitutions, and an HC-CDR3 comprisingthe amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or avariant thereof comprising up to about 3 (for example about any of 1, 2,or 3) amino acid substitutions; and ii) a light chain variable domaincomprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, and b) an effectormolecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W(SEQ ID NO: 87), an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and an HC-CDR3 comprising theamino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii)a light chain variable domain comprising an LC-CDR1 comprising the aminoacid sequence of S/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), and anLC-CDR3 comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQID NO: 121); wherein X can be any amino acid, and b) an effectormolecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, an HC-CDR2 comprising the amino acid sequenceof any one of SEQ ID NOs: 67-76, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86, or a variant thereof comprising up to about 5(such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; andii) a light chain variable domain comprising an LC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 90-99, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, an LC-CDR2 comprising the amino acid sequenceof any one of SEQ ID NOs: 100-109, or a variant thereof comprising up toabout 3 (such as about any of 1, 2, or 3) amino acid substitutions, andan LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:110-119, or a variant thereof comprising up to about 5 (such as aboutany of 1, 2, 3, 4, or 5) amino acid substitutions, and b) an effectormolecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66; anHC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:67-76; and an HC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 77-86; or a variant thereof comprising up to about 5 (suchas about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRsequences; and ii) a light chain variable domain sequence comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99; an LC-CDR2 comprising the amino acid sequence of any one of SEQID NOs: 100-109; and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119; or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutionsin the LC-CDR sequences, and b) an effector molecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisingi) a heavy chain variable domain sequence comprising an HC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 57-66; anHC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:67-76; and an HC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 77-86; and ii) a light chain variable domain sequencecomprising an LC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 100-109; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119, and b) an effector molecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisinga heavy chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 17-26, or a variant thereof having at least about 95%(for example at least about any of 96%, 97%, 98%, or 99%) sequenceidentity, and a light chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 27-36, or a variant thereof having atleast about 95% (for example at least about any of 96%, 97%, 98%, or99%) sequence identity, and b) an effector molecule.

In some embodiments, there is provided an anti-AMC immunoconjugatecomprising a) an anti-AMC antibody moiety that specifically binds to acomplex comprising an AFP peptide and an MHC class I protein comprisinga heavy chain variable domain comprising the amino acid sequence of anyone of SEQ ID NOs: 17-26 and a light chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 27-36, b) an effectormolecule.

Nucleic Acids

Nucleic acid molecules encoding the anti-AMC constructs or anti-AMCantibody moieties are also contemplated. In some embodiments, there isprovided a nucleic acid (or a set of nucleic acids) encoding afull-length anti-AMC antibody. In some embodiments, there is provided anucleic acid (or a set of nucleic acids) encoding a multi-specificanti-AMC molecule (e.g., a multi-specific anti-AMC antibody, abispecific anti-AMC antibody, or a bispecific T-cell engager anti-AMCantibody), or polypeptide portion thereof. In some embodiments, there isprovided a nucleic acid (or a set of nucleic acids) encoding an anti-AMCCAR. In some embodiments, there is provided a nucleic acid (or a set ofnucleic acids) encoding an anti-AMC immunoconjugate, or polypeptideportion thereof.

The present application also includes variants to these nucleic acidsequences. For example, the variants include nucleotide sequences thathybridize to the nucleic acid sequences encoding the anti-AMC constructsor anti-AMC antibody moieties of the present application under at leastmoderately stringent hybridization conditions.

The present invention also provides vectors in which a nucleic acid ofthe present invention is inserted.

In brief summary, the expression of an anti-AMC construct (e.g.,anti-AMC CAR) or polypeptide portion thereof by a natural or syntheticnucleic acid encoding the anti-AMC construct or polypeptide portionthereof can be achieved by inserting the nucleic acid into anappropriate expression vector, such that the nucleic acid is operablylinked to 5′ and 3′ regulatory elements, including for example apromoter (e.g., a lymphocyte-specific promoter) and a 3′ untranslatedregion (UTR). The vectors can be suitable for replication andintegration in eukaryotic host cells. Typical cloning and expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The nucleic acids of the present invention may also be used for nucleicacid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In some embodiments, the inventionprovides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers (see, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In someembodiments, lentivirus vectors are used. Vectors derived fromretroviruses such as the lentivirus are suitable tools to achievelong-term gene transfer since they allow long-term, stable integrationof a transgene and its propagation in daughter cells. Lentiviral vectorshave the added advantage over vectors derived from onco-retrovirusessuch as murine leukemia viruses in that they can transducenon-proliferating cells, such as hepatocytes. They also have the addedadvantage of low immunogenicity.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell can also containeither a selectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother aspects, the selectable marker may be carried on a separate pieceof DNA and used in a co-transfection procedure. Both selectable markersand reporter genes may be flanked with appropriate regulatory sequencesto enable expression in the host cells. Useful selectable markersinclude, for example, antibiotic-resistance genes, such as neo and thelike.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,β-galactosidase, chloramphenicol acetyl transferase, secreted alkalinephosphatase, or the green fluorescent protein gene (e.g., Ui-Tel et al.,2000 FEBS Letters 479: 79-82). Suitable expression systems are wellknown and may be prepared using known techniques or obtainedcommercially. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). In some embodiments, the introduction of a polynucleotideinto a host cell is carried out by calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodof inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virus1, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

AFP and MHC Class I Proteins

Alpha-fetoprotein (AFP, α-fetoprotein; also referred to asalpha-1-fetoprotein, alpha-fetoglobulin, or alpha fetal protein) is a591 amino acid glycoprotein that in humans is encoded by the AFP gene.The AFP gene is located on the q arm of chromosome 4 (4q25). AFP is amajor plasma protein produced by the yolk sac and the liver during fetaldevelopment. It is thought to be the fetal form of serum albumin. AFPbinds to copper, nickel, fatty acids and bilirubin, and is found inmonomeric, dimeric and trimeric forms. AFP is the most abundant plasmaprotein found in the human fetus. Plasma levels decrease rapidly afterbirth but begin decreasing prenatally starting at the end of the firsttrimester. Normal adult levels are usually achieved by the age of 8 to12 months. The function of AFP in adult humans is unknown; however, inrodents it binds estradiol to prevent the transport of this hormoneacross the placenta to the fetus. The main function of this is toprevent the virilization of female fetuses. As human AFP does not bindestrogen, its function in humans is less clear.

Some of the diseases in which AFP will be elevated in a person include,for example, hepatocellular carcinoma/hepatoma, germ cell tumor,metastatic liver cancer, omphalocele, neural tube defects, yolk sactumors, and ataxia telangiectasia.

MHC class I proteins are one of two primary classes of majorhistocompatibility complex (MHC) molecules (the other being MHC classII) and are found on nearly every nucleated cell of the body. Theirfunction is to display fragments of proteins from within the cell to Tcells; healthy cells will be ignored, while cells containing foreignproteins will be attacked by the immune system. Because MHC class Iproteins present peptides derived from cytosolic proteins, the pathwayof MHC class I presentation is often called the cytosolic or endogenouspathway. Class I MHC molecules bind peptides generated mainly fromdegradation of cytosolic proteins by the proteasome. The MHC I:peptidecomplex is then inserted into the plasma membrane of the cell. Thepeptide is bound to the extracellular part of the class I MHC molecule.Thus, the function of the class I MHC is to display intracellularproteins to cytotoxic T cells (CTLs). However, class I MHC can alsopresent peptides generated from exogenous proteins, in a process knownas cross-presentation.

MHC class I proteins consist of two polypeptide chains, a andβ2-microglobulin (132M). The two chains are linked noncovalently viainteraction of b2m and the α3 domain. Only the α chain is polymorphicand encoded by a HLA gene, while the b2m subunit is not polymorphic andencoded by the β-2 microglobulin gene. The α3 domain is plasmamembrane-spanning and interacts with the CD8 co-receptor of T-cells. Theα3-CD8 interaction holds the MHC I molecule in place while the T cellreceptor (TCR) on the surface of the cytotoxic T cell binds its α1-α2heterodimer ligand, and checks the coupled peptide for antigenicity. Theα1 and α2 domains fold to make up a groove for peptides to bind. MHCclass I proteins bind peptides that are 8-10 amino acid in length.

The human leukocyte antigen (HLA) genes are the human versions of theMHC genes. The three major MHC class I proteins in humans are HLA-A,HLA-B, and HLA-C, while the 3 minor ones are HLA-E, HLA-F, and HLA-G.HLA-A is ranked among the genes in humans with the fastest-evolvingcoding sequence. As of December 2013, there were 2432 known HLA-Aalleles coding for 1740 active proteins and 117 null proteins. The HLA-Agene is located on the short arm of chromosome 6 and encodes the larger,α-chain, constituent of HLA-A. Variation of HLA-A α-chain is key to HLAfunction. This variation promotes genetic diversity in the population.Since each HLA has a different affinity for peptides of certainstructures, greater variety of HLAs means greater variety of antigens tobe ‘presented’ on the cell surface, enhancing the likelihood that asubset of the population will be resistant to any given foreign invader.This decreases the likelihood that a single pathogen has the capabilityto wipe out the entire human population. Each individual can express upto two types of HLA-A, one from each of their parents. Some individualswill inherit the same HLA-A from both parents, decreasing theirindividual HLA diversity; however, the majority of individuals willreceive two different copies of HLA-A. This same pattern follows for allHLA groups. In other words, a person can only express either one or twoof the 2432 known HLA-A alleles.

All alleles receive at least a four digit classification, e.g.,HLA-A*02:12. The A signifies which HLA gene the allele belongs to. Thereare many HLA-A alleles, so that classification by serotype simplifiescategorization. The next pair of digits indicates this assignment. Forexample, HLA-A*02:02, HLA-A*02:04, and HLA-A*02:324 are all members ofthe A2 serotype (designated by the *02 prefix). This group is theprimary factor responsible for HLA compatibility. All numbers after thiscannot be determined by serotyping and are designated through genesequencing. The second set of digits indicates what HLA protein isproduced. These are assigned in order of discovery and as of December2013 there are 456 different HLA-A02 proteins known (assigned namesHLA-A*02:01 to HLA-A*02:456). The shortest possible HLA name includesboth of these details. Each extension beyond that signifies a nucleotidechange that may or may not change the protein.

In some embodiments, the anti-AMC antibody moiety specifically binds toa complex comprising an AFP peptide and an MHC class I protein, whereinthe MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.In some embodiments, the MHC class I protein is HLA-A, HLA-B, or HLA-C.In some embodiments, the MHC class I protein is HLA-A. In someembodiments, the MHC class I protein is HLA-B. In some embodiments, theMHC class I protein is HLA-C. In some embodiments, the MHC class Iprotein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11,HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30,HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68,HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class Iprotein is HLA-A02. In some embodiments, the MHC class I protein is anyone of HLA-A*02:01-555, such as HLA-A*02:01, HLA-A*02:02, HLA-A*02:03,HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08,HLA-A*02:09, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13,HLA-A*02:14, HLA-A*02:15, HLA-A*02:16, HLA-A*02:17, HLA-A*02:18,HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, or HLA-A*02:24. Insome embodiments, the MHC class I protein is HLA-A*02:01. HLA-A*02:01 isexpressed in 39-46% of all Caucasians, and therefore represents asuitable choice of MHC class I protein for use in the present invention.

AFP peptides suitable for use in generating anti-AMC antibody moietiescan be determined, for example, based on the presence ofHLA-A*02:01-binding motifs and cleavage sites for proteasomes andimmune-proteasomes using computer prediction models known to those ofskill in the art. For predicting MHC binding sites, such models include,but are not limited to, IEDB (Vita et al., The immune epitope database(IEDB) 3.0. Nucleic Acids Res. 2014 Oct. 9. pii: gku938), ProPred1(described in more detail in Singh and Raghava, ProPred: prediction ofHLA-DR binding sites. BIOINFORMATICS 17(12):1236-1237, 2001), andSYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching andT-Cell Epitope Prediction. in Immunoinformatics Methods in MolecularBiology, vol 409(1): 75-93, 2007).

Once appropriate peptides have been identified, peptide synthesis may bedone in accordance with protocols well known to those of skill in theart. Because of their relatively small size, the peptides of theinvention may be directly synthesized in solution or on a solid supportin accordance with conventional peptide synthesis techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. The synthesis of peptides in solutionphase has become a well-established procedure for large-scale productionof synthetic peptides and as such is a suitable alternative method ofpreparing the peptides of the invention (See for example, Solid PhasePeptide Synthesis by John Morrow Stewart and Martin et al. Applicationof Almez-mediated Amidation Reactions to Solution Phase PeptideSynthesis, Tetrahedron Letters Vol. 39, pages 1517-1520, 1998).

The binding activity of candidate AFP peptides can be tested using theantigen-processing-deficient T2 cell line, which increases expression ofHLA-A when stabilized by a peptide in the antigen-presenting groove. T2cells are pulsed with the candidate peptide for a time sufficient tostabilize HLA-A expression on the cell surface, which can be measuredusing any methods known in the art, such as by immunostaining with afluorescently labeled monoclonal antibody specific for HLA-A (forexample, BB7.2) followed by fluorescence-activated cell-sorting (FACS)analysis.

Preparation of Anti-AMC Antibodies and Anti-AMC Antibody Moieties

In some embodiments, the anti-AMC antibody or anti-AMC antibody moietyis a monoclonal antibody. Monoclonal antibodies can be prepared, e.g.,using hybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975) and Sergeeva et al., Blood, 117(16):4262-4272,using the phage display methods described herein and in the Examplesbelow, or using recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).

In a hybridoma method, a hamster, mouse, or other appropriate hostanimal is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro. The immunizing agent can includea polypeptide or a fusion protein of the protein of interest, or acomplex comprising at least two molecules, such as a complex comprisingan AFP peptide and an MHC class I protein. Generally, peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell. See, e.g., Goding, Monoclonal Antibodies: Principlesand Practice (New York: Academic Press, 1986), pp. 59-103. Immortalizedcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Usually, rat or mouse myelomacell lines are employed. The hybridoma cells can be cultured in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, immortalized cells.For example, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which prevents the growth of HGPRT-deficientcells.

In some embodiments, the immortalized cell lines fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. In some embodiments, the immortalized cell lines are murinemyeloma lines, which can be obtained, for instance, from the SalkInstitute Cell Distribution Center, San Diego, Calif. and the AmericanType Culture Collection, Manassas, Va. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al. Monoclonal Antibody Production Techniques andApplications (Marcel Dekker, Inc.: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide. The binding specificity of monoclonal antibodies producedby the hybridoma cells can be determined by immunoprecipitation or by anin vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.Goding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the sub clones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The anti-AMC antibodies or antibody moieties may also be identified byscreening combinatorial libraries for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are reviewed, e.g., in Hoogenboom et al., Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., 2001) and further described, e.g., in McCafferty et al., Nature348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods inMolecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J.,2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

The antibodies or antigen-binding fragments thereof can be preparedusing phage display to screen libraries for antibodies specific to acomplex comprising an AFP peptide and an MHC class I protein. Thelibrary can be a human scFv phage display library having a diversity ofat least one×10⁹ (such as at least about any of 1×10⁹, 2.5×10⁹, 5×10⁹,7.5×10⁹, 1×10¹⁰, 2.5×10¹⁰, 5×10¹⁰, 7.5×10¹⁰, or 1×10¹¹) unique humanantibody fragments. In some embodiments, the library is a naïve humanlibrary constructed from DNA extracted from human PMBCs and spleens fromhealthy donors, encompassing all human heavy and light chainsubfamilies. In some embodiments, the library is a naïve human libraryconstructed from DNA extracted from PBMCs isolated from patients withvarious diseases, such as patients with autoimmune diseases, cancerpatients, and patients with infectious diseases. In some embodiments,the library is a semi-synthetic human library, wherein heavy chain CDR3is completely randomized, with all amino acids (with the exception ofcysteine) equally likely to be present at any given position (see, e.g.,Hoet, R. M. et al., Nat. Biotechnol. 23(3):344-348, 2005). In someembodiments, the heavy chain CDR3 of the semi-synthetic human libraryhas a length from about 5 to about 24 (such as about any of 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) aminoacids. In some embodiments, the library is a non-human phage displaylibrary.

Phage clones that bind to the AMC with high affinity can be selected byiterative binding of phage to the AMC, which is bound to a solid support(such as, for example, beads for solution panning or mammalian cells forcell panning), followed by removal of non-bound phage and by elution ofspecifically bound phage. In an example of solution panning, the AMC canbe biotinylated for immobilization to a solid support. The biotinylatedAMC is mixed with the phage library and a solid support, such asstreptavidin-conjugated Dynabeads M-280, and then AMC-phage-beadcomplexes are isolated. The bound phage clones are then eluted and usedto infect an appropriate host cell, such as E. coli XL1-Blue, forexpression and purification. In an example of cell panning, T2 cells (aTAP-deficient, HLA-A*02:01+ lymphoblast cell line) loaded with the AFPpeptide of the AMC are mixed with the phage library, after which thecells are collected and the bound clones are eluted and used to infectan appropriate host cell for expression and purification. The panningcan be performed for multiple (such as about any of 2, 3, 4, 5, 6 ormore) rounds with either solution panning, cell panning, or acombination of both, to enrich for phage clones binding specifically tothe AMC. Enriched phage clones can be tested for specific binding to theAMC by any methods known in the art, including for example ELISA andFACS.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Hybridoma cells asdescribed above or AMC-specific phage clones of the invention can serveas a source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such assimian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also can be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains and/orframework regions in place of the homologous non-human sequences (U.S.Pat. No. 4,816,567; Morrison et al., supra) or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a nonimmunoglobulin polypeptide. Such a nonimmunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies can be monovalent antibodies. Methods for preparingmonovalent antibodies are known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using any method known in the art.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. In some embodiments, the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding is present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology, 121: 210 (1986).

Human and Humanized Antibodies

The anti-AMC antibodies or antibody moieties can be humanized antibodiesor human antibodies. Humanized forms of non-human (e.g., murine)antibodies are chimeric immunoglobulins, immunoglobulin chains, orfragments thereof (such as Fv, Fab, Fab′, F(ab′)₂, scFv, or otherantigen-binding subsequences of antibodies) that typically containminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a CDR of the recipient are replaced by residues from a CDRof a non-human species (donor antibody) such as mouse, rat, or rabbithaving the desired specificity, affinity, and capacity. In someinstances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Humanized antibodies canalso comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences. In general, thehumanized antibody can comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin, andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. In some embodiments, the humanizedantibody will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. See, e.g., Joneset al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. According to someembodiments, humanization can be essentially performed following themethod of Winter and co-workers (Jones et al., Nature, 321: 522-525(1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al.,Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO97/17852. Alternatively, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed thatclosely resembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275) or by using varioustechniques known in the art, including phage display libraries.Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner etal. are also available for the preparation of human monoclonalantibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1): 86-95(1991).

Multi-Specific Antibodies

In some embodiments, the anti-AMC construct is a multi-specificantibody. Suitable methods for making multi-specific (e.g., bispecific)antibodies are well known in the art. For example, the production ofbispecific antibodies can based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two pairs eachhave different specificities, and upon association result in aheterodimeric antibody (see, e.g., Milstein and Cuello, Nature, 305:537-539 (1983); WO 93/08829, and Traunecker et al., EMBO J. 10: 3655(1991)). Because of the random assortment of immunoglobulin heavy andlight chains, these hybridomas (quadromas) produce a potential mixtureof ten different antibody molecules, of which only one has the correctbispecific structure. The purification of the correct molecule isusually accomplished by affinity chromatography steps. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO,10: 3655-3659 (1991). Alternatively, the combining of heavy and lightchains can be directed by taking advantage of species-restricted pairing(see, e.g., Lindhofer et al., J. Immunol., 155:219-225 (1995)) and thepairing of heavy chains can be directed by use of “knob-into hole”engineering of CH3 domains (see, e.g., U.S. Pat. No. 5,731,168; Ridgwayet al., Protein Eng., 9(7):617-621 (1996)). Multi-specific antibodiesmay also be made by engineering electrostatic steering effects formaking antibody Fc-heterodimeric molecules (see, e.g., WO2009/089004A1). In yet another method, stable bispecific antibodies canbe generated by controlled Fab-arm exchange, where two parentalantibodies having distinct antigen specificity and matched pointmutations in the CH3 domains are mixed in reducing condition to allowfor separation, reassembly, and reoxidation to form highly purebispecific antibodies. Labrigin et al., Proc. Natl. Acad. Sci.,110(13):5145-5150 (2013). Such antibodies, comprising a mixture ofheavy-chain/light-chain pairs, are also referred to herein as“heteromultimeric antibodies”.

Antibodies or antigen-binding fragments thereof having differentspecificities can also be chemically cross-linked to generatemulti-specific heteroconjugate antibodies. For example, two F(ab′)2molecules, each having specificity for a different antigen, can bechemically linked. Pullarkat et al., Trends Biotechnol., 48:9-21 (1999).Such antibodies have, for example, been proposed to target immune-systemcells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment ofHIV infection. WO 91/00360; WO 92/200373; EP 03089. It is contemplatedthat the antibodies can be prepared in vitro using known methods insynthetic protein chemistry, including those involving crosslinkingagents. For example, immunotoxins can be constructed using adisulfide-exchange reaction or by forming a thioether bond. Examples ofsuitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.Pat. No. 4,676,980.

In some embodiments, multi-specific antibodies can be prepared usingrecombinant DNA techniques. For example, a bispecific antibody can beengineered by fusing two scFvs, such as by fusing them through a peptidelinker, resulting in a tandem scFv. One example of a tandem scFv is abispecific T cell engager. Bispecific T cell engagers are made bylinking an anti-CD3 scFv to an scFv specific for a surface antigen of atarget cell, such as a tumor-associated antigen (TAA), resulting in theredirection of T cells to the target cells. Mack et al., Proc. Natl.Acad. Sci., 92:7021-7025 (1995); Brischwein et al., Mol. Immunol.,43(8):1129-1143 (2006). By shortening the length of a peptide linkerbetween two variable domains, they can be prevented from self-assemblingand forced to pair with domains on a second polypeptide, resulting in acompact bispecific antibody called a diabody (Db). Holliger et al.,Proc. Natl. Acad. Sci., 90:6444-6448 (1993). The two polypeptides of aDb each comprise a VH connected to a VL by a linker which is too shortto allow pairing between the two domains on the same chain. Accordingly,the VH and VL domains of one polypeptide are forced to pair with thecomplementary VL and VH domains of another polypeptide, thereby formingtwo antigen-binding sites. In a modification of this format, the twopolypeptides are linked by another peptide linker, resulting in a singlechain diabody (scDb). In yet another modification of the Db format,dual-affinity retargeting (DART) bispecific antibodies can be generatedby introducing a disulfide linkage between cysteine residues at theC-terminus of each polypeptide, optionally including domains prior tothe C-terminal cysteine residues that drive assembly of the desiredheterodimeric structure. Veri et al., Arthritis Rheum., 62(7):1933-1943(2010). Dual-variable-domain immunoglobulins (DVD-Ig™), in which thetarget-binding variable domains of two monoclonal antibodies arecombined via naturally occurring linkers to yield a tetravalent,bispecific antibody, are also known in the art. Gu and Ghayur, MethodsEnzymol., 502:25-41 (2012). In yet another format, Dock and Lock (DNL),bispecific antibodies are prepared by taking advantage of thedimerization of a peptide (DDD2) derived from the regulatory subunit ofhuman cAMP-dependent protein kinase (PKA) with a peptide (AD2) derivedfrom the anchoring domains of human A kinase anchor proteins (AKAPs).Rossi et al., Proc. Natl. Acad. Sci., 103:6841-6846 (2006).

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).This method can also be utilized for the production of antibodyhomodimers.

Anti-AMC Variants

In some embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

In some embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

Conservative substitutions are shown in Table 5 below.

TABLE 5 CONSERVATIVE SUBSTITITIONS Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped into different classes according to commonside-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c. acidic: Asp, Glu;

d. basic: His, Lys, Arg;

e. residues that influence chain orientation: Gly, Pro;

f aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

An exemplary substitutional variant is an affinity matured antibody,which may be conveniently generated, e.g., using phage display-basedaffinity maturation techniques. Briefly, one or more CDR residues aremutated and the variant antibodies displayed on phage and screened for aparticular biological activity (e.g. binding affinity). Alterations(e.g., substitutions) may be made in HVRs, e.g., to improve antibodyaffinity. Such alterations may be made in HVR “hotspots,” i.e., residuesencoded by codons that undergo mutation at high frequency during thesomatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.207:179-196 (2008)), and/or specificity determining residues (SDRs),with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).)

In some embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In some embodiments, substitutions, insertions, or deletions may occurwithin one or more HVRs so long as such alterations do not substantiallyreduce the ability of the antibody to bind antigen. For example,conservative alterations (e.g., conservative substitutions as providedherein) that do not substantially reduce binding affinity may be made inHVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In someembodiments of the variant VH and VL sequences provided above, each HVReither is unaltered, or contains no more than one, two or three aminoacid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex can bedetermined to identify contact points between the antibody and antigen.Such contact residues and neighboring residues may be targeted oreliminated as candidates for substitution. Variants may be screened todetermine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

Fc Region Variants

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of a full-length anti-AMC antibodyprovided herein, thereby generating an Fc region variant. In someembodiments, the Fc region variant has enhanced antibody dependentcellular cytotoxicity (ADCC) effector function, often related to bindingto Fc receptors (FcRs). In some embodiments, the Fc region variant hasdecreased ADCC effector function. There are many examples of changes ormutations to Fc sequences that can alter effector function. For example,WO 00/42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001)describe antibody variants with improved or diminished binding to FcRs.The contents of those publications are specifically incorporated hereinby reference.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism ofaction of therapeutic antibodies against tumor cells. ADCC is acell-mediated immune defense whereby an effector cell of the immunesystem actively lyses a target cell (e.g., a cancer cell), whosemembrane-surface antigens have been bound by specific antibodies (e.g.,an anti-AMC antibody). The typical ADCC involves activation of NK cellsby antibodies. An NK cell expresses CD16 which is an Fc receptor. Thisreceptor recognizes, and binds to, the Fc portion of an antibody boundto the surface of a target cell. The most common Fc receptor on thesurface of an NK cell is called CD16 or FcγRIII. Binding of the Fcreceptor to the Fc region of an antibody results in NK cell activation,release of cytolytic granules and consequent target cell apoptosis. Thecontribution of ADCC to tumor cell killing can be measured with aspecific test that uses NK-92 cells that have been transfected with ahigh-affinity FcR. Results are compared to wild-type NK-92 cells that donot express the FcR.

In some embodiments, the invention contemplates an anti-AMC constructvariant comprising an FC region that possesses some but not all effectorfunctions, which makes it a desirable candidate for applications inwhich the half-life of the anti-AMC construct in vivo is important yetcertain effector functions (such as CDC and ADCC) are unnecessary ordeleterious. In vitro and/or in vivo cytotoxicity assays can beconducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express FcγRIII only, whereasmonocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assay methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, there is provided an anti-AMC construct (e.g., afull-length anti-AMC antibody) variant comprising a variant Fc regioncomprising one or more amino acid substitutions which improve ADCC. Insome embodiments, the variant Fc region comprises one or more amino acidsubstitutions which improve ADCC, wherein the substitutions are atpositions 298, 333, and/or 334 of the variant Fc region (EU numbering ofresidues). In some embodiments, the anti-AMC construct (e.g.,full-length anti-AMC antibody) variant comprises the following aminoacid substitution in its variant Fc region: S298A, E333A, and K334A.

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

In some embodiments, there is provided an anti-AMC construct (e.g., afull-length anti-AMC antibody) variant comprising a variant Fc regioncomprising one or more amino acid substitutions which increase half-lifeand/or improve binding to the neonatal Fc receptor (FcRn). Antibodieswith increased half-lives and improved binding to FcRn are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

Anti-AMC constructs (such as full-length anti-AMC antibodies) comprisingany of the Fc variants described herein, or combinations thereof, arecontemplated.

Glycosylation Variants

In some embodiments, an anti-AMC construct provided herein is altered toincrease or decrease the extent to which the anti-AMC construct isglycosylated. Addition or deletion of glycosylation sites to an anti-AMCconstruct may be conveniently accomplished by altering the amino acidsequence of the anti-AMC construct or polypeptide portion thereof suchthat one or more glycosylation sites is created or removed.

Where the anti-AMC construct comprises an Fc region, the carbohydrateattached thereto may be altered. Native antibodies produced by mammaliancells typically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an anti-AMC construct of the invention may be made inorder to create anti-AMC construct variants with certain improvedproperties.

In some embodiments, anti-AMC construct (such as full-length anti-AMCantibody) variants are provided comprising an Fc region wherein acarbohydrate structure attached to the Fc region has reduced fucose orlacks fucose, which may improve ADCC function. Specifically, anti-AMCconstructs are contemplated herein that have reduced fusose relative tothe amount of fucose on the same anti-AMC construct produced in awild-type CHO cell. That is, they are characterized by having a loweramount of fucose than they would otherwise have if produced by nativeCHO cells (e.g., a CHO cell that produce a native glycosylation pattern,such as, a CHO cell containing a native FUT8 gene). In some embodiments,the anti-AMC construct is one wherein less than about 50%, 40%, 30%,20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. Forexample, the amount of fucose in such an anti-AMC construct may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. In someembodiments, the anti-AMC construct is one wherein none of the N-linkedglycans thereon comprise fucose, i.e., wherein the anti-AMC construct iscompletely without fucose, or has no fucose or is afucosylated. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchasα-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Anti-AMC construct (such as full-length anti-AMC antibody) variants arefurther provided with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region of the anti-AMCconstruct is bisected by GlcNAc. Such anti-AMC construct (such asfull-length anti-AMC antibody) variants may have reduced fucosylationand/or improved ADCC function. Examples of such antibody variants aredescribed, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara etal., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Anti-AMCconstruct (such as full-length anti-AMC antibody) variants with at leastone galactose residue in the oligosaccharide attached to the Fc regionare also provided. Such anti-AMC construct variants may have improvedCDC function. Such antibody variants are described, e.g., in WO1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764(Raju, S.).

In some embodiments, the anti-AMC construct (such as full-lengthanti-AMC antibody) variants comprising an Fc region are capable ofbinding to an FcγRIII. In some embodiments, the anti-AMC construct (suchas full-length anti-AMC antibody) variants comprising an Fc region haveADCC activity in the presence of human effector cells or have increasedADCC activity in the presence of human effector cells compared to theotherwise same anti-AMC construct (such as full-length anti-AMCantibody) comprising a human wild-type IgG1Fc region.

Cysteine Engineered Variants

In some embodiments, it may be desirable to create cysteine engineeredanti-AMC constructs (such as full-length anti-AMC antibodies) in whichone or more amino acid residues are substituted with cysteine residues.In some embodiments, the substituted residues occur at accessible sitesof the anti-AMC construct. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theanti-AMC construct and may be used to conjugate the anti-AMC constructto other moieties, such as drug moieties or linker-drug moieties, tocreate an anti-AMC immunoconjugate, as described further herein.Cysteine engineered anti-AMC constructs (such as full-length anti-AMCantibodies) may be generated as described, e.g., in U.S. Pat. No.7,521,541.

Derivatives

In some embodiments, an anti-AMC construct provided herein may befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the anti-AMC construct include but are not limited towater soluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the anti-AMC construct may vary, and if more than onepolymer are attached, they can be the same or different molecules. Ingeneral, the number and/or type of polymers used for derivatization canbe determined based on considerations including, but not limited to, theparticular properties or functions of the anti-AMC construct to beimproved, whether the anti-AMC construct derivative will be used in atherapy under defined conditions, etc.

In some embodiments, conjugates of an anti-AMC construct andnonproteinaceous moiety that may be selectively heated by exposure toradiation are provided. In some embodiments, the nonproteinaceous moietyis a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength, andincludes, but is not limited to, wavelengths that do not harm ordinarycells, but which heat the nonproteinaceous moiety to a temperature atwhich cells proximal to the anti-AMC construct-nonproteinaceous moietyare killed.

CAR Effector Cell Preparation

The present invention in one aspect provides effector cells (such aslymphocytes, for example T cells) expressing an anti-AMC CAR. Exemplarymethods of preparing effector cells (such as T cells) expressing theanti-AMC CARs (anti-AMC CAR effector cells, such as anti-AMC CAR Tcells) are provided herein.

In some embodiments, an anti-AMC CAR effector cell (such as T cell) canbe generated by introducing a vector (including for example a lentiviralvector) comprising an anti-AMC CAR (for example a CAR comprising ananti-AMC antibody moiety and CD28 and CD3ζ intracellular signalingsequences) into the effector cell (such as T cell). In some embodiments,the anti-AMC CAR effector cells (such as T cells) of the invention areable to replicate in vivo, resulting in long-term persistence that canlead to sustained control of an AFP-positive disease (such as cancer,e.g., HCC).

In some embodiments, the invention relates to administering agenetically modified T cell expressing an anti-AMC CAR for the treatmentof a patient having an AFP-positive disease or at risk of having anAFP-positive disease using lymphocyte infusion. In some embodiments,autologous lymphocyte infusion is used in the treatment. AutologousPBMCs are collected from a patient in need of treatment and T cells areactivated and expanded using the methods described herein and known inthe art and then infused back into the patient.

In some embodiments, the anti-AMC CAR T cell expresses an anti-AMC CARcomprising an anti-AMC antibody moiety (also referred to herein as an“anti-AMC CAR T cell”). In some embodiments, the anti-AMC CAR T cellexpresses an anti-AMC CAR comprising an extracellular domain comprisingan anti-AMC antibody moiety and an intracellular domain comprisingintracellular signaling sequences of CD3ζ and CD28. The anti-AMC CARTcells of the invention can undergo robust in vivo T cell expansion andcan establish AMC-specific memory cells that persist at high levels foran extended amount of time in blood and bone marrow. In someembodiments, the anti-AMC CAR T cells of the invention infused into apatient can eliminate AMC-presenting cells, such as AMC-presentingcancer cells, in vivo in patients having an AFP-positive disease. Insome embodiments, the anti-AMC CAR T cells of the invention infused intoa patient can eliminate AMC-presenting cells, such as AMC-presentingcancer cells, in vivo in patients having an AFP-positive disease that isrefractory to at least one conventional treatment.

In some embodiments, the anti-AMC CAR T cell expresses the anti-AMC CARwith even cell surface distribution. Even cell surface distribution canbe characterized, for example, by staining patterns with continuousappearance and even thickness or signal intensity. For example, in someembodiments, a composition, such as a pharmaceutical composition,comprising anti-AMC CAR T cells comprises fewer than about 10% (such asfewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) cells withaggregation of the anti-AMC CAR on the cell surface. Aggregation can becharacterized, for example, by staining patterns with uneven thicknessor signal intensity, or discontinuous, lumpy, punctate, and/or unevendistribution patterns. In some embodiments, the anti-AMC CAR T cellexpresses the anti-AMC CAR with less than about 10% (such as less thanabout 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) aggregation of the anti-AMC CARon the cell surface. In some embodiments, the anti-AMC CAR T cell has alow level of antigen-independent anti-AMC CAR activation. In someembodiments, the anti-AMC CART cell has a low level of T cellexhaustion. T cell exhaustion naturally occurs during conditions ofextended immune activation, such as with cancer or chronic infection,where T cells become dysfunctional. T cell exhaustion may becharacterized by impaired effector function, prolonged expression ofinhibitory receptors, and/or an altered transcriptional state comparedto functional effector or memory T cells. Optimal clearance of tumorcells and infections is prevented by T cell exhaustion. T cellexhaustion of the anti-AMC CAR T cell can be characterized by any meansknown in the art, for example, by determining its functional and/orphenotypic profile (Wherry, E. J., Nature immunology 12(6): 492-499,2011; Jiang, Y., et al., Cell death & disease 6(6): e1792, 2015). Forexample, in some embodiments, the anti-AMC CAR T cell expresses lowlevels of one or more markers of T cell exhaustion, including, forexample, PD-1, LAG-3, TIM-3, CTLA-4, BTLA, and TIGIT. In someembodiments, the anti-AMC CAR T cell maintains levels characteristic ofnon-exhausted T cells for IL-2 production, TNF-α production, IFN-γproduction, and granzyme B production, and/or maintains ex vivo killingcapacity in the presence of target cells, suggesting that the anti-AMCCART cell is not undergoing self-activation and premature exhaustion.

Prior to expansion and genetic modification of the T cells, a source ofT cells is obtained from a subject. T cells can be obtained from anumber of sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Insome embodiments of the present invention, any number of T cell linesavailable in the art may be used. In some embodiments of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In some embodiments, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In some embodiments, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In some embodiments, the cells are washed with phosphate buffered saline(PBS). In some embodiments, the wash solution lacks calcium and may lackmagnesium or may lack many if not all divalent cations. As those ofordinary skill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such asCa²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other saline solutions withor without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In some embodiments, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in some embodiments, T cells are isolated by incubationwith anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such asDYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positiveselection of the desired T cells. In some embodiments, the time periodis about 30 minutes. In some embodiments, the time period ranges from 30minutes to 36 hours or longer and all integer values there between. Insome embodiments, the time period is at least one, 2, 3, 4, 5, or 6hours. In some embodiments, the time period is 10 to 24 hours. In someembodiments, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such as in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immune-compromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8⁺ T cells. Thus, by simply shortening orlengthening the time T cells are allowed to bind to the CD3/CD28 beadsand/or by increasing or decreasing the ratio of beads to T cells,subpopulations of T cells can be preferentially selected for or againstat culture initiation or at other time points during the process.Additionally, by increasing or decreasing the ratio of anti-CD3 and/oranti-CD28 antibodies on the beads or other surface, subpopulations of Tcells can be preferentially selected for or against at cultureinitiation or at other desired time points. The skilled artisan wouldrecognize that multiple rounds of selection can also be used in thecontext of this invention. In some embodiments, it may be desirable toperform the selection procedure and use the “unselected” cells in theactivation and expansion process. “Unselected” cells can also besubjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD 14, CD20, CD11b, CD 16,HLA-DR, and CD8. In some embodiments, it may be desirable to enrich foror positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62Lhi, GITR⁺, and FoxP3⁺. Alternatively, in someembodiments, T regulatory cells are depleted by anti-CD25 conjugatedbeads or other similar methods of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In some embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in some embodiments, aconcentration of about 2 billion cells/ml is used. In some embodiments,a concentration of about 1 billion cells/ml is used. In someembodiments, greater than about 100 million cells/ml is used. In someembodiments, a concentration of cells of about any of 10, 15, 20, 25,30, 35, 40, 45, or 50 million cells/ml is used. In some embodiments, aconcentration of cells of about any of 75, 80, 85, 90, 95, or 100million cells/ml is used. In some embodiments, a concentration of about125 or about 150 million cells/ml is used. Using high concentrations canresult in increased cell yield, cell activation, and cell expansion.Further, use of high cell concentrations allows more efficient captureof cells that may weakly express target antigens of interest, such asCD28-negative T cells, or from samples where there are many tumor cellspresent (i.e., leukemic blood, tumor tissue, etc.). Such populations ofcells may have therapeutic value and would be desirable to obtain. Forexample, using high concentration of cells allows more efficientselection of CD8⁺ T cells that normally have weaker CD28 expression.

In some embodiments of the present invention, T cells are obtained froma patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, insome embodiments, mobilization (for example, mobilization with GM-CSF)and conditioning regimens can be used to create a condition in a subjectwherein repopulation, recirculation, regeneration, and/or expansion ofparticular cell types is favored, especially during a defined window oftime following therapy. Illustrative cell types include T cells, Bcells, dendritic cells, and other cells of the immune system.

Whether prior to or after genetic modification of the T cells to expressa desirable anti-AMC CAR, the T cells can be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4⁺T cells or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody.Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone,Besancon, France) can be used as can other methods commonly known in theart (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al.,J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1-2):53-63, 1999).

Immunoconjugate Preparation

The anti-AMC immunoconjugates may be prepared using any methods known inthe art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. PatentApplication Publication No. 2014322129, incorporated by reference hereinin their entirety.

The anti-AMC antibody moiety of an anti-AMC immunoconjugate may be“attached to” the effector molecule by any means by which the anti-AMCantibody moiety can be associated with, or linked to, the effectormolecule. For example, the anti-AMC antibody moiety of an anti-AMCimmunoconjugate may be attached to the effector molecule by chemical orrecombinant means. Chemical means for preparing fusions or conjugatesare known in the art and can be used to prepare the anti-AMCimmunoconjugate. The method used to conjugate the anti-AMC antibodymoiety and effector molecule must be capable of joining the bindingprotein with the effector molecule without interfering with the abilityof the binding protein to bind to the antigen on the target cell.

The anti-AMC antibody moiety of an anti-AMC immunoconjugate may belinked indirectly to the effector molecule. For example, the anti-AMCantibody moiety of an anti-AMC immunoconjugate may be directly linked toa liposome containing the effector molecule of one of several types. Theeffector molecule(s) and/or the anti-AMC antibody moiety may also bebound to a solid surface.

In some embodiments, the anti-AMC antibody moiety of an anti-AMCimmunoconjugate and the effector molecule are both proteins and can beconjugated using techniques well known in the art. There are severalhundred crosslinkers available that can conjugate two proteins. (See forexample “Chemistry of Protein Conjugation and Crosslinking”. 1991, ShansWong, CRC Press, Ann Arbor). The crosslinker is generally chosen basedon the reactive functional groups available or inserted on the anti-AMCantibody moiety and/or effector molecule. In addition, if there are noreactive groups, a photoactivatible crosslinker can be used. In certaininstances, it may be desirable to include a spacer between the anti-AMCantibody moiety and the effector molecule. Crosslinking agents known tothe art include the homobifunctional agents: glutaraldehyde,dimethyladipimidate and Bis(diazobenzidine) and the heterobifunctionalagents: m Maleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

In some embodiments, the anti-AMC antibody moiety of an anti-AMCimmunoconjugate may be engineered with specific residues for chemicalattachment of the effector molecule. Specific residues used for chemicalattachment of molecule known to the art include lysine and cysteine. Thecrosslinker is chosen based on the reactive functional groups insertedon the anti-AMC antibody moiety, and available on the effector molecule.

An anti-AMC immunoconjugate may also be prepared using recombinant DNAtechniques. In such a case a DNA sequence encoding the anti-AMC antibodymoiety is fused to a DNA sequence encoding the effector molecule,resulting in a chimeric DNA molecule. The chimeric DNA sequence istransfected into a host cell that expresses the fusion protein. Thefusion protein can be recovered from the cell culture and purified usingtechniques known in the art.

Examples of attaching an effector molecule, which is a label, to thebinding protein include the methods described in Hunter, et al., Nature144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, etal., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. andCytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging AndRadioimmunotherapy, Elsevier, N.Y. (1983); and Colcher et al., “Use OfMonoclonal Antibodies As Radiopharmaceuticals For The Localization OfHuman Carcinoma Xenografts In Athymic Mice”, Meth. Enzymol., 121:802-16(1986).

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as ⁹⁹Tc or ¹²³I, ¹⁸⁶Re, ¹⁸⁸Re and ¹¹¹In can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys.Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Immunoconjugates of the antibody moiety and a cytotoxic agent may bemade using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetnaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.The linker may be a “cleavable linker” facilitating release of thecytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The anti-AMC immunoconjugates of the invention expressly contemplate,but are not limited to, ADC prepared with cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

Pharmaceutical Compositions

Also provided herein are compositions (such as pharmaceuticalcompositions, also referred to herein as formulations) comprising ananti-AMC construct. In some embodiments, the composition furthercomprises a cell (such as an effector cell, e.g., a T cell) associatedwith the anti-AMC construct. In some embodiments, there is provided apharmaceutical composition comprising an anti-AMC construct and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition further comprises a cell (such as an effectorcell, e.g., a T cell) associated with the anti-AMC construct.

Suitable formulations of the anti-AMC constructs are obtained by mixingan anti-AMC construct having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propylparaben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such asolyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG). Exemplary formulationsare described in WO98/56418, expressly incorporated herein by reference.Lyophilized formulations adapted for subcutaneous administration aredescribed in WO97/04801. Such lyophilized formulations may bereconstituted with a suitable diluent to a high protein concentrationand the reconstituted formulation may be administered subcutaneously tothe individual to be treated herein. Lipofectins or liposomes can beused to deliver the anti-AMC constructs of this invention into cells.

The formulation herein may also contain one or more active compounds inaddition to the anti-AMC construct as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. For example, it may bedesirable to further provide an anti-neoplastic agent, a growthinhibitory agent, a cytotoxic agent, or a chemotherapeutic agent inaddition to the anti-AMC construct. Such molecules are suitably presentin combination in amounts that are effective for the purpose intended.The effective amount of such other agents depends on the amount ofanti-AMC construct present in the formulation, the type of disease ordisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asdescribed herein or about from 1 to 99% of the heretofore employeddosages.

The anti-AMC constructs may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Sustained-release preparations may be prepared.

Sustained-release preparations of the anti-AMC constructs can beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody (or fragment thereof), which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydro gelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they can denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization of anti-AMC constructsdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S-S bond formation throughthio-disulfide interchange, stabilization can be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

In some embodiments, the anti-AMC construct is formulated in a buffercomprising a citrate, NaCl, acetate, succinate, glycine, polysorbate 80(Tween 80), or any combination of the foregoing. In some embodiments,the anti-AMC construct is formulated in a buffer comprising about 100 mMto about 150 mM glycine. In some embodiments, the anti-AMC construct isformulated in a buffer comprising about 50 mM to about 100 mM NaCl. Insome embodiments, the anti-AMC construct is formulated in a buffercomprising about 10 mM to about 50 mM acetate. In some embodiments, theanti-AMC construct is formulated in a buffer comprising about 10 mM toabout 50 mM succinate. In some embodiments, the anti-AMC construct isformulated in a buffer comprising about 0.005% to about 0.02%polysorbate 80. In some embodiments, the anti-AMC construct isformulated in a buffer having a pH between about 5.1 and 5.6. In someembodiments, the anti-AMC construct is formulated in a buffer comprising10 mM citrate, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80,wherein the formulation is at pH 5.5.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, e.g., filtration through sterilefiltration membranes.

Methods for Treatment Using Anti-AMC Constructs

The anti-AMC constructs and/or compositions of the invention can beadministered to individuals (e.g., mammals such as humans) to treat adisease and/or disorder involving abnormally high AFP expression (alsoreferred to herein as an “AFP-positive” disease or disorder), including,for example, cancer (such as hepatocellular carcinoma, germ cell tumor,and breast cancer). The present application thus in some embodimentsprovides a method of treating an AFP-positive disease (such as cancer)in an individual comprising administering to the individual an effectiveamount of a composition (such as a pharmaceutical composition)comprising an anti-AMC construct comprising an anti-AMC antibody moiety,such as any one of the anti-AMC constructs described herein. In someembodiments, the composition further comprises a cell (such as aneffector cell) associated with the anti-AMC construct. In someembodiments, the cancer is selected, for example, from the groupconsisting of hepatocellular carcinoma, germ cell tumor, and breastcancer. In some embodiments, the cancer is hepatocellular carcinoma. Insome embodiments, the cancer is hepatocellular carcinoma and thetreating comprises preventing the spread of the cancer, e.g., inhibiting(such as preventing) metastasis of the cancer. In some embodiments, thecancer is metastatic hepatocellular carcinoma. In some embodiments, theindividual is human.

For example, in some embodiments, there is provided a method of treatingan AFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein. Insome embodiments, the AFP peptide is AFP158 (SEQ ID NO: 4). In someembodiments, the MHC class I protein is HLA-A02. In some embodiments,the MHC class I protein is HLA-A*02:01. In some embodiments, theanti-AMC construct is non-naturally occurring. In some embodiments, theanti-AMC construct is a full-length antibody. In some embodiments, theanti-AMC construct is a multi-specific (such as bispecific) molecule. Insome embodiments, the anti-AMC construct is a chimeric antigen receptor.In some embodiments, the anti-AMC construct is an immunoconjugate. Insome embodiments, the composition further comprises a cell (such as aneffector cell) associated with the anti-AMC construct. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP158 peptide (SEQ ID NO: 4) andHLA-A*02:01. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, thecomposition further comprises a cell (such as an effector cell)associated with the anti-AMC construct. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, the individualis human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein,wherein the anti-AMC antibody moiety comprises: i) a heavy chainvariable domain sequence comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or avariant thereof comprising up to about 3 (for example about any of 1, 2,or 3) amino acid substitutions, an HC-CDR2 comprising the amino acidsequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid. In some embodiments, theanti-AMC construct is non-naturally occurring. In some embodiments, theanti-AMC construct is a full-length antibody. In some embodiments, theanti-AMC construct is a multi-specific (such as bispecific) molecule. Insome embodiments, the anti-AMC construct is a chimeric antigen receptor.In some embodiments, the anti-AMC construct is an immunoconjugate. Insome embodiments, the composition further comprises a cell (such as aneffector cell) associated with the anti-AMC construct. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein,wherein the anti-AMC antibody moiety comprises: i) a heavy chainvariable domain sequence comprising an HC-CDR1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), anHC-CDR2 comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQID NO: 88), and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); and ii) a light chain variabledomain comprising an LC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid. In some embodiments, the anti-AMCconstruct is non-naturally occurring. In some embodiments, the anti-AMCconstruct is a full-length antibody. In some embodiments, the anti-AMCconstruct is a multi-specific (such as bispecific) molecule. In someembodiments, the anti-AMC construct is a chimeric antigen receptor. Insome embodiments, the anti-AMC construct is an immunoconjugate. In someembodiments, the composition further comprises a cell (such as aneffector cell) associated with the anti-AMC construct. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein,wherein the anti-AMC antibody moiety comprises: i) a heavy chainvariable domain sequence comprising an HC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 57-66, or a variant thereofcomprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)amino acid substitutions; an HC-CDR2 comprising the amino acid sequenceof any one of SEQ ID NOs: 67-76, or a variant thereof comprising up toabout 5 (for example about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; and an HC-CDR3 comprising the amino acid sequence of anyone of SEQ ID NOs: 77-86; or a variant thereof comprising up to about 5(for example about any of 1, 2, 3, 4, or 5) amino acid substitutions;and ii) a light chain variable domain sequence comprising an LC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 90-99, or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions; an LC-CDR2 comprising the aminoacid sequence of any one of SEQ ID NOs: 100-109, or a variant thereofcomprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)amino acid substitutions; and an LC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 110-119; or a variant thereofcomprising up to about 5 (for example about any of 1, 2, 3, 4, or 5)amino acid substitutions. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, thecomposition further comprises a cell (such as an effector cell)associated with the anti-AMC construct. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, the individualis human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein,wherein the anti-AMC antibody moiety comprises: i) a heavy chainvariable domain sequence comprising an HC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 57-66; an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76; and an HC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 77-86; or avariant thereof comprising up to about 5 (for example about any of 1, 2,3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and ii) alight chain variable domain sequence comprising an LC-CDR1 comprisingthe amino acid sequence of any one of SEQ ID NOs: 90-99; an LC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 100-109;and an LC-CDR3 comprising the amino acid sequence of any one of SEQ IDNOs: 110-119; or a variant thereof comprising up to about 5 (for exampleabout any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDRsequences. In some embodiments, the anti-AMC construct is non-naturallyoccurring. In some embodiments, the anti-AMC construct is a full-lengthantibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, thecomposition further comprises a cell (such as an effector cell)associated with the anti-AMC construct. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, the individualis human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MHC class I protein,wherein the anti-AMC antibody moiety comprises: i) a heavy chainvariable domain sequence comprising an HC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 57-66; an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76; and an HC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 77-86; andii) a light chain variable domain sequence comprising an LC-CDR1comprising the amino acid sequence of any one of SEQ ID NOs: 90-99; anLC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs:100-109; and an LC-CDR3 comprising the amino acid sequence of any one ofSEQ ID NOs: 110-119. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, thecomposition further comprises a cell (such as an effector cell)associated with the anti-AMC construct. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, the individualis human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MCH class I protein,wherein the anti-AMC antibody moiety comprises a heavy chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:17-26, or a variant thereof having at least about 95% (for example atleast about any of 96%, 97%, 98%, or 99%) sequence identity, and a lightchain variable domain comprising the amino acid sequence of any one ofSEQ ID NOs: 27-36, or a variant thereof having at least about 95% (forexample at least about any of 96%, 97%, 98%, or 99%) sequence identity.In some embodiments, the anti-AMC construct is non-naturally occurring.In some embodiments, the anti-AMC construct is a full-length antibody.In some embodiments, the anti-AMC construct is a multi-specific (such asbispecific) molecule. In some embodiments, the anti-AMC construct is achimeric antigen receptor. In some embodiments, the anti-AMC constructis an immunoconjugate. In some embodiments, the composition furthercomprises a cell (such as an effector cell) associated with the anti-AMCconstruct. In some embodiments, the AFP-positive disease is cancer. Insome embodiments, the cancer is, for example, hepatocellular carcinoma,germ cell tumor, or breast cancer. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma and the treating comprises preventing thespread of the cancer, e.g., inhibiting (such as preventing) metastasisof the cancer. In some embodiments, the cancer is metastatichepatocellular carcinoma. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an anti-AMCconstruct comprising an anti-AMC antibody moiety that specifically bindsto a complex comprising an AFP peptide and an MCH class I protein,wherein the anti-AMC antibody moiety comprises a heavy chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:17-26 and a light chain variable domain comprising the amino acidsequence of any one of SEQ ID NOs: 27-36. In some embodiments, theanti-AMC construct is non-naturally occurring. In some embodiments, theanti-AMC construct is a full-length antibody. In some embodiments, theanti-AMC construct is a multi-specific (such as bispecific) molecule. Insome embodiments, the anti-AMC construct is a chimeric antigen receptor.In some embodiments, the anti-AMC construct is an immunoconjugate. Insome embodiments, the composition further comprises a cell (such as aneffector cell) associated with the anti-AMC construct. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating metastatichepatocellular carcinoma in an individual comprising administering tothe individual an effective amount of a composition comprising ananti-AMC construct according to any of the embodiments described above.In some embodiments, the individual is human.

In some embodiments, there is provided a method of inhibiting (such aspreventing) metastasis of hepatocellular carcinoma in an individualcomprising administering to the individual an effective amount of acomposition comprising an anti-AMC construct according to any of theembodiments described above. In some embodiments, the individual ishuman.

In some embodiments of any of the methods for treating an AFP-positivedisease described above, the anti-AMC construct is conjugated to a cell(such as an immune cell, e.g., a T cell) prior to being administered tothe individual. Thus, for example, there is provided a method oftreating an AFP-positive disease in an individual comprising a)conjugating any one of the anti-AMC constructs described herein to acell (such as an immune cell, e.g., a T cell) to form an anti-AMCconstruct/cell conjugate, and b) administering to the individual aneffective amount of a composition comprising the anti-AMC construct/cellconjugate. In some embodiments, the cell is derived from the individual.In some embodiments, the cell is not derived from the individual. Insome embodiments, the anti-AMC construct is conjugated to the cell bycovalent linkage to a molecule on the surface of the cell. In someembodiments, the anti-AMC construct is conjugated to the cell bynon-covalent linkage to a molecule on the surface of the cell. In someembodiments, the anti-AMC construct is conjugated to the cell byinsertion of a portion of the anti-AMC construct into the outer membraneof the cell. In some embodiments, the anti-AMC construct isnon-naturally occurring. In some embodiments, the anti-AMC construct isa full-length antibody. In some embodiments, the anti-AMC construct is amulti-specific (such as bispecific) molecule. In some embodiments, theanti-AMC construct is a chimeric antigen receptor. In some embodiments,the anti-AMC construct is an immunoconjugate. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, the individualis human.

In some embodiments, the individual is a mammal (e.g., human, non-humanprimate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). Insome embodiments, the individual is a human. In some embodiments, theindividual is a clinical patient, a clinical trial volunteer, anexperimental animal, etc. In some embodiments, the individual is youngerthan about 60 years old (including for example younger than about any of50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, theindividual is older than about 60 years old (including for example olderthan about any of 70, 80, 90, or 100 years old). In some embodiments,the individual is diagnosed with or genetically prone to one or more ofthe diseases or disorders described herein (such as hepatocellularcarcinoma, germ cell tumor, and breast cancer). In some embodiments, theindividual has one or more risk factors associated with one or morediseases or disorders described herein.

The present application in some embodiments provides a method ofdelivering an anti-AMC construct (such as any one of the anti-AMCconstructs described herein) to a cell presenting on its surface acomplex comprising an AFP peptide and an MHC class I protein in anindividual, the method comprising administering to the individual acomposition comprising the anti-AMC construct. In some embodiments, theanti-AMC construct to be delivered is associated with a cell (such as aneffector cell, e.g., a T cell).

Many diagnostic methods for cancer (such as hepatocellular carcinoma,germ cell tumor, and breast cancer) or any other disease exhibitingabnormal AFP expression and the clinical delineation of those diseasesare known in the art. Such methods include, but are not limited to,e.g., immunohistochemistry, PCR, and fluorescent in situ hybridization(FISH).

In some embodiments, the anti-AMC constructs and/or compositions of theinvention are administered in combination with a second, third, orfourth agent (including, e.g., an antineoplastic agent, a growthinhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) totreat diseases or disorders involving abnormal AFP expression. In someembodiments, the anti-AMC construct is administered in combination withan agent that increases the expression of MHC class I proteins and/orenhances the surface presentation of AFP peptides by MHC class Iproteins. In some embodiments, the agent includes, for example, IFNreceptor agonists, Hsp90 inhibitors, enhancers of p53 expression, andchemotherapeutic agents. In some embodiments, the agent is an IFNreceptor agonist including, for example, IFNγ, IFNβ, and IFNα. In someembodiments, the agent is an Hsp90 inhibitor including, for example,tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspimycin (IPI-504),IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305), PU-H71,Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387,SNX-5422, DS-2248, and XL888. In some embodiments, the agent is anenhancer of p53 expression including, for example, 5-fluorouracil andnutlin-3. In some embodiments, the agent is a chemotherapeutic agentincluding, for example, topotecan, etoposide, cisplatin, paclitaxel, andvinblastine.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual, wherein the cells expressing AFPdo not normally present, or present at relatively low levels, a complexcomprising an AFP protein and an MHC class I protein on their surface(such as germ cell tumor cells), the method comprising administering tothe individual a composition comprising an anti-AMC construct incombination with an agent that increases the expression of MHC class Iproteins and/or enhances the surface presentation of AFP peptides by MHCclass I proteins. In some embodiments, the agent includes, for example,IFN receptor agonists, Hsp90 inhibitors, enhancers of p53 expression,and chemotherapeutic agents. In some embodiments, the agent is an IFNreceptor agonist including, for example, IFNγ, IFNβ, and IFNα. In someembodiments, the agent is an Hsp90 inhibitor including, for example,tanespimycin (17-AAG), alvespimycin (17-DMAG), retaspimycin (IPI-504),IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932 (CUDC-305), PU-H71,Ganetespib (STA-9090), NVP-AUY922 (VER-52269), HSP990, KW-2478, AT13387,SNX-5422, DS-2248, and XL888. In some embodiments, the agent is anenhancer of p53 expression including, for example, 5-fluorouracil andnutlin-3. In some embodiments, the agent is a chemotherapeutic agentincluding, for example, topotecan, etoposide, cisplatin, paclitaxel, andvinblastine.

Cancer treatments can be evaluated by, e.g., tumor regression, tumorweight or size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,quality of life, protein expression and/or activity. Approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of response through radiological imaging.

In some embodiments, the efficacy of treatment is measured as thepercentage tumor growth inhibition (% TGI), calculated using theequation 100−(T/C×100), where T is the mean relative tumor volume of thetreated tumor, and C is the mean relative tumor volume of a non-treatedtumor. In some embodiments, the % TGI is about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, or more than 95%.

Dosing and Method of Administering the Anti-AMC Construct Compositions

The dose of the anti-AMC construct compositions administered to anindividual (such as a human) may vary with the particular composition,the mode of administration, and the type of disease being treated. Insome embodiments, the amount of the composition is effective to resultin an objective response (such as a partial response or a completeresponse). In some embodiments, the amount of the anti-AMC constructcomposition is sufficient to result in a complete response in theindividual. In some embodiments, the amount of the anti-AMC constructcomposition is sufficient to result in a partial response in theindividual. In some embodiments, the amount of the anti-AMC constructcomposition administered (for example when administered alone) issufficient to produce an overall response rate of more than about any of20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%,85%, or 90% among a population of individuals treated with the anti-AMCconstruct composition. Responses of an individual to the treatment ofthe methods described herein can be determined, for example, based onRECIST levels.

In some embodiments, the amount of the composition is sufficient toprolong progress-free survival of the individual. In some embodiments,the amount of the composition is sufficient to prolong overall survivalof the individual. In some embodiments, the amount of the composition(for example when administered along) is sufficient to produce clinicalbenefit of more than about any of 50%, 60%, 70%, or 77% among apopulation of individuals treated with the anti-AMC constructcomposition.

In some embodiments, the amount of the composition, alone or incombination with a second, third, and/or fourth agent, is an amountsufficient to decrease the size of a tumor, decrease the number ofcancer cells, or decrease the growth rate of a tumor by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% comparedto the corresponding tumor size, number of cancer cells, or tumor growthrate in the same subject prior to treatment or compared to thecorresponding activity in other subjects not receiving the treatment.Standard methods can be used to measure the magnitude of this effect,such as in vitro assays with purified enzyme, cell-based assays, animalmodels, or human testing.

In some embodiments, the amount of the anti-AMC construct (e.g.,full-length anti-AMC antibody, multi-specific anti-AMC molecule,anti-AMC CAR, or anti-AMC immunoconjugate) in the composition is belowthe level that induces a toxicological effect (i.e., an effect above aclinically acceptable level of toxicity) or is at a level where apotential side effect can be controlled or tolerated when thecomposition is administered to the individual.

In some embodiments, the amount of the composition is close to a maximumtolerated dose (MTD) of the composition following the same dosingregimen. In some embodiments, the amount of the composition is more thanabout any of 80%, 90%, 95%, or 98% of the MTD.

In some embodiments, the amount of an anti-AMC construct (e.g.,full-length anti-AMC antibody, multi-specific anti-AMC molecule,anti-AMC CAR, or anti-AMC immunoconjugate) in the composition isincluded in a range of about 0.001 pg to about 1000 μg.

In some embodiments of any of the above aspects, the effective amount ofan anti-AMC construct (e.g., full-length anti-AMC antibody,multi-specific anti-AMC molecule, anti-AMC CAR, or anti-AMCimmunoconjugate) in the composition is in the range of about 0.1 pg/kgto about 100 mg/kg of total body weight.

The anti-AMC construct compositions can be administered to an individual(such as human) via various routes, including, for example, intravenous,intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, transmucosal, and transdermal. In someembodiments, sustained continuous release formulation of the compositionmay be used. In some embodiments, the composition is administeredintravenously. In some embodiments, the composition is administeredintraportally. In some embodiments, the composition is administeredintraarterially. In some embodiments, the composition is administeredintraperitoneally. In some embodiments, the composition is administeredintrahepatically. In some embodiments, the composition is administeredby hepatic arterial infusion.

Anti-AMC CAR Effector Cell Therapy

The present application also provides methods of using an anti-AMC CARto redirect the specificity of an effector cell (such as a primary Tcell) to a complex comprising an AFP peptide and an MHC class I protein.Thus, the present invention also provides a method of stimulating aneffector cell-mediated response (such as a T cell-mediated immuneresponse) to a target cell population or tissue comprisingAMC-presenting cells in a mammal, comprising the step of administeringto the mammal an effector cell (such as a T cell) that expresses ananti-AMC CAR.

Anti-AMC CAR effector cells (such as T cells) expressing the anti-AMCCAR can be infused to a recipient in need thereof. The infused cell isable to kill AMC-presenting cells in the recipient. In some embodiments,unlike antibody therapies, anti-AMC CAR effector cells (such as T cells)are able to replicate in vivo resulting in long-term persistence thatcan lead to sustained tumor control.

In some embodiments, the anti-AMC CAR effector cells are anti-AMC CAR Tcells that can undergo robust in vivo T cell expansion and can persistfor an extended amount of time. In some embodiments, the anti-AMC CAR Tcells of the invention develop into specific memory T cells that can bereactivated to inhibit any additional tumor formation or growth.

The anti-AMC CART cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal. Insome embodiments, the mammal is a human.

With respect to ex vivo immunization, of least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding ananti-AMC CAR to the cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing an anti-AMC CAR disclosed herein. Theanti-AMC CAR cell can be administered to a mammalian recipient toprovide a therapeutic benefit. The mammalian recipient may be a humanand the anti-AMC CAR cell can be autologous with respect to therecipient. Alternatively, the cells can be allogeneic, syngeneic orxenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34⁺ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

The anti-AMC CAR effector cells (such as T cells) of the presentinvention may be administered either alone, or as a pharmaceuticalcomposition in combination with diluents and/or with other componentssuch as IL-2 or other cytokines or cell populations. Briefly,pharmaceutical compositions of the present invention may compriseanti-AMC CAR effector cells (such as T cells), in combination with oneor more pharmaceutically or physiologically acceptable carriers,diluents or excipients. Such compositions may comprise buffers such asneutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. In some embodiments, anti-AMC CAReffector cell (such as T cell) compositions are formulated forintravenous administration.

The precise amount of the anti-AMC CAR effector cell (such as T cell)compositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). In some embodiments, apharmaceutical composition comprising the anti-AMC CAR effector cells(such as T cells) is administered at a dosage of about 10⁴ to about 10⁹cells/kg body weight, such any of about 10⁴ to about 10⁵, about 10⁵ toabout 10⁶, about 10⁶ to about 10⁷, about 10⁷ to about 10⁸, or about 10⁸to about 10⁹ cells/kg body weight, including all integer values withinthose ranges. Anti-AMC CAR effect cell (such as T cell) compositions mayalso be administered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regimen for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In some embodiments, it may be desired to administer activated anti-AMCCAR T cells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In some embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In some embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc.

The administration of the anti-AMC CAR effector cells (such as T cells)may be carried out in any convenient manner, including by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i.v.)injection, or intraperitoneally. In some embodiments, the anti-AMC CAReffector cell (such as T cell) compositions of the present invention areadministered to a patient by intradermal or subcutaneous injection. Insome embodiments, the anti-AMC CAR effector cell (such as T cell)compositions of the present invention are administered by i.v.injection. The compositions of anti-AMC CAR effector cells (such as Tcells) may be injected directly into a tumor, lymph node, or site ofinfection.

Thus, for example, in some embodiments, there is provided a method oftreating an AFP-positive disease in an individual comprisingadministering to the individual an effective amount of a compositioncomprising an effector cell (such as a T cell) expressing an anti-AMCCAR comprising a) an extracellular domain comprising an anti-AMCantibody moiety that specifically binds to a complex comprising an AFPpeptide and an MHC class I protein, b) a transmembrane domain, and c) anintracellular signaling domain comprising a CD3ζ intracellular signalingsequence and a CD28 intracellular signaling sequence. In someembodiments, the AFP peptide is AFP158 (SEQ ID NO: 4). In someembodiments, the MHC class I protein is HLA-A02. In some embodiments,the MHC class I protein is HLA-A*02:01. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, b) a transmembranedomain, and c) an intracellular signaling domain comprising a CD3ζintracellular signaling sequence and a CD28 intracellular signalingsequence. In some embodiments, the AFP-positive disease is cancer. Insome embodiments, the cancer is, for example, hepatocellular carcinoma,germ cell tumor, or breast cancer. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma and the treating comprises preventing thespread of the cancer, e.g., inhibiting (such as preventing) metastasisof the cancer. In some embodiments, the cancer is metastatichepatocellular carcinoma. In some embodiments, the administration is viaintravenous, intraperitoneal, or intratumoral route. In someembodiments, the administration is via intravenous route. In someembodiments, the administration is via intratumoral route. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, b) a transmembrane domain, and c)an intracellular signaling domain comprising a CD3ζ intracellularsignaling sequence and a CD28 intracellular signaling sequence. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the administration is via intravenous, intraperitoneal, orintratumoral route. In some embodiments, the administration is viaintravenous route. In some embodiments, the administration is viaintratumoral route. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingan HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence. In some embodiments, theAFP-positive disease is cancer. In some embodiments, the cancer is, forexample, hepatocellular carcinoma, germ cell tumor, or breast cancer. Insome embodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the HC-CDR sequences; and ii) a light chainvariable domain sequence comprising an LC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 110-119; ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions in the LC-CDR sequences; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence. In some embodiments, the AFP-positivedisease is cancer. In some embodiments, the cancer is, for example,hepatocellular carcinoma, germ cell tumor, or breast cancer. In someembodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence. In some embodiments, the AFP-positivedisease is cancer. In some embodiments, the cancer is, for example,hepatocellular carcinoma, germ cell tumor, or breast cancer. In someembodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 17-26, or a variantthereof having at least about 95% (for example at least about any of96%, 97%, 98%, or 99%) sequence identity, and a light chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:27-36, or a variant thereof having at least about 95% sequence identity;b) a transmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence. In some embodiments, the AFP-positivedisease is cancer. In some embodiments, the cancer is, for example,hepatocellular carcinoma, germ cell tumor, or breast cancer. In someembodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26 and a light chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 27-36; b) a transmembrane domain, and c) an intracellular signalingdomain comprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence. In some embodiments, the AFP-positivedisease is cancer. In some embodiments, the cancer is, for example,hepatocellular carcinoma, germ cell tumor, or breast cancer. In someembodiments, the cancer is hepatocellular carcinoma. In someembodiments, the cancer is hepatocellular carcinoma and the treatingcomprises preventing the spread of the cancer, e.g., inhibiting (such aspreventing) metastasis of the cancer. In some embodiments, the cancer ismetastatic hepatocellular carcinoma. In some embodiments, theadministration is via intravenous, intraperitoneal, or intratumoralroute. In some embodiments, the administration is via intravenous route.In some embodiments, the administration is via intratumoral route. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein, b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence, wherein the administeringcomprises local injection of the composition at an injection site distalto a site of the AFP-positive disease (such as an AFP-positive tumor).In some embodiments, the injection site is a first AFP-positive tumor(i.e., the administration is via intratumoral route). In someembodiments, the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the injection site is a firstAFP-positive tumor and the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the AFP peptide is AFP158 (SEQID NO: 4). In some embodiments, the MHC class I protein is HLA-A02. Insome embodiments, the MHC class I protein is HLA-A*02:01. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP158 peptide (SEQ ID NO:4) and HLA-A*02:01, b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence, wherein the administeringcomprises local injection of the composition at an injection site distalto a site of the AFP-positive disease (such as an AFP-positive tumor).In some embodiments, the injection site is a first AFP-positive tumor(i.e., the administration is via intratumoral route). In someembodiments, the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the injection site is a firstAFP-positive tumor and the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the AFP-positive disease iscancer. In some embodiments, the cancer is, for example, hepatocellularcarcinoma, germ cell tumor, or breast cancer. In some embodiments, thecancer is hepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma and the treating comprises preventing thespread of the cancer, e.g., inhibiting (such as preventing) metastasisof the cancer. In some embodiments, the cancer is metastatichepatocellular carcinoma. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, an HC-CDR2 comprising the amino acid sequence ofI/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), or a variant thereofcomprising up to about 3 (for example about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofQ-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO: 121), or a variant thereof comprisingup to about 3 (for example about any of 1, 2, or 3) amino acidsubstitutions; wherein X can be any amino acid, b) a transmembranedomain, and c) an intracellular signaling domain comprising a CD3ζintracellular signaling sequence and a CD28 intracellular signalingsequence, wherein the administering comprises local injection of thecomposition at an injection site distal to a site of the AFP-positivedisease (such as an AFP-positive tumor). In some embodiments, theinjection site is a first AFP-positive tumor (i.e., the administrationis via intratumoral route). In some embodiments, the site of theAFP-positive disease is a second AFP-positive tumor. In someembodiments, the injection site is a first AFP-positive tumor and thesite of the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence ofG-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), an HC-CDR2 comprisingthe amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQ ID NO: 88), and anHC-CDR3 comprising the amino acid sequence of A/G-X-W/Y-Y-X-X-X-F/Y-D(SEQ ID NO: 89); and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence ofS/T-G/S-D/N-I/V-A/G-A/S/V-X-H/Y (SEQ ID NO: 120), and an LC-CDR3comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQ ID NO:121); wherein X can be any amino acid, b) a transmembrane domain, and c)an intracellular signaling domain comprising a CD3ζ intracellularsignaling sequence and a CD28 intracellular signaling sequence, whereinthe administering comprises local injection of the composition at aninjection site distal to a site of the AFP-positive disease (such as anAFP-positive tumor). In some embodiments, the injection site is a firstAFP-positive tumor (i.e., the administration is via intratumoral route).In some embodiments, the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the injection site is a firstAFP-positive tumor and the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the AFP-positive disease iscancer. In some embodiments, the cancer is, for example, hepatocellularcarcinoma, germ cell tumor, or breast cancer. In some embodiments, thecancer is hepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma and the treating comprises preventing thespread of the cancer, e.g., inhibiting (such as preventing) metastasisof the cancer. In some embodiments, the cancer is metastatichepatocellular carcinoma. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingan HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:57-66, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 67-76, or a variantthereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5)amino acid substitutions, and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86, or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions; and ii) a light chain variable domain comprising anLC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs:90-99, or a variant thereof comprising up to about 5 (such as about anyof 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109, or a variantthereof comprising up to about 3 (such as about any of 1, 2, or 3) aminoacid substitutions, and an LC-CDR3 comprising the amino acid sequence ofany one of SEQ ID NOs: 110-119, or a variant thereof comprising up toabout 5 (such as about any of 1, 2, 3, 4, or 5) amino acidsubstitutions; b) a transmembrane domain, and c) an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence, wherein the administeringcomprises local injection of the composition at an injection site distalto a site of the AFP-positive disease (such as an AFP-positive tumor).In some embodiments, the injection site is a first AFP-positive tumor(i.e., the administration is via intratumoral route). In someembodiments, the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the injection site is a firstAFP-positive tumor and the site of the AFP-positive disease is a secondAFP-positive tumor. In some embodiments, the AFP-positive disease iscancer. In some embodiments, the cancer is, for example, hepatocellularcarcinoma, germ cell tumor, or breast cancer. In some embodiments, thecancer is hepatocellular carcinoma and the treating comprises preventingthe spread of the cancer, e.g., inhibiting (such as preventing)metastasis of the cancer. In some embodiments, the cancer is metastatichepatocellular carcinoma. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; or a variant thereofcomprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) aminoacid substitutions in the HC-CDR sequences; and ii) a light chainvariable domain sequence comprising an LC-CDR1 comprising the amino acidsequence of any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising theamino acid sequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3comprising the amino acid sequence of any one of SEQ ID NOs: 110-119; ora variant thereof comprising up to about 5 (such as about any of 1, 2,3, 4, or 5) amino acid substitutions in the LC-CDR sequences; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence, wherein the administering compriseslocal injection of the composition at an injection site distal to a siteof the AFP-positive disease (such as an AFP-positive tumor). In someembodiments, the injection site is a first AFP-positive tumor (i.e., theadministration is via intratumoral route). In some embodiments, the siteof the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the injection site is a first AFP-positive tumor and thesite of the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain sequencecomprising an HC-CDR1 comprising the amino acid sequence of any one ofSEQ ID NOs: 57-66; an HC-CDR2 comprising the amino acid sequence of anyone of SEQ ID NOs: 67-76; and an HC-CDR3 comprising the amino acidsequence of any one of SEQ ID NOs: 77-86; and ii) a light chain variabledomain sequence comprising an LC-CDR1 comprising the amino acid sequenceof any one of SEQ ID NOs: 90-99; an LC-CDR2 comprising the amino acidsequence of any one of SEQ ID NOs: 100-109; and an LC-CDR3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 110-119; b) atransmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence, wherein the administering compriseslocal injection of the composition at an injection site distal to a siteof the AFP-positive disease (such as an AFP-positive tumor). In someembodiments, the injection site is a first AFP-positive tumor (i.e., theadministration is via intratumoral route). In some embodiments, the siteof the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the injection site is a first AFP-positive tumor and thesite of the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising i) a heavy chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 17-26, or a variantthereof having at least about 95% (for example at least about any of96%, 97%, 98%, or 99%) sequence identity, and a light chain variabledomain comprising the amino acid sequence of any one of SEQ ID NOs:27-36, or a variant thereof having at least about 95% sequence identity;b) a transmembrane domain, and c) an intracellular signaling domaincomprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence, wherein the administering compriseslocal injection of the composition at an injection site distal to a siteof the AFP-positive disease (such as an AFP-positive tumor). In someembodiments, the injection site is a first AFP-positive tumor (i.e., theadministration is via intratumoral route). In some embodiments, the siteof the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the injection site is a first AFP-positive tumor and thesite of the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating anAFP-positive disease in an individual comprising administering to theindividual an effective amount of a composition comprising an effectorcell (such as a T cell) expressing an anti-AMC CAR comprising a) anextracellular domain comprising an anti-AMC antibody moiety thatspecifically binds to a complex comprising an AFP peptide and an MHCclass I protein comprising a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26 and a light chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 27-36; b) a transmembrane domain, and c) an intracellular signalingdomain comprising a CD3ζ intracellular signaling sequence and a CD28intracellular signaling sequence, wherein the administering compriseslocal injection of the composition at an injection site distal to a siteof the AFP-positive disease (such as an AFP-positive tumor). In someembodiments, the injection site is a first AFP-positive tumor (i.e., theadministration is via intratumoral route). In some embodiments, the siteof the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the injection site is a first AFP-positive tumor and thesite of the AFP-positive disease is a second AFP-positive tumor. In someembodiments, the AFP-positive disease is cancer. In some embodiments,the cancer is, for example, hepatocellular carcinoma, germ cell tumor,or breast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is hepatocellular carcinomaand the treating comprises preventing the spread of the cancer, e.g.,inhibiting (such as preventing) metastasis of the cancer. In someembodiments, the cancer is metastatic hepatocellular carcinoma. In someembodiments, the individual is human.

In some embodiments, there is provided a method of treating metastatichepatocellular carcinoma in an individual comprising administering tothe individual an effective amount of a composition comprising aneffector cell (such as a T cell) expressing an anti-AMC CAR according toany of the embodiments described above. In some embodiments, theindividual is human.

In some embodiments, there is provided a method of inhibiting (such aspreventing) metastasis of hepatocellular carcinoma in an individualcomprising administering to the individual an effective amount of acomposition comprising an effector cell (such as a T cell) expressing ananti-AMC CAR according to any of the embodiments described above. Insome embodiments, the individual is human.

In some embodiments, there is provided a method of priming T cells in anindividual comprising administering to the individual an effectiveamount of a composition comprising an effector cell (such as a T cell)expressing an anti-AMC CAR according to any of the anti-AMC CARsdescribed above. In some embodiments, the individual has an AFP-positivedisease. In some embodiments, the AFP-positive disease is cancer. Insome embodiments, the cancer is, for example, hepatocellular carcinoma,germ cell tumor, or breast cancer. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the administration is viaintravenous, intraperitoneal, or intratumoral route. In someembodiments, the administration is via intravenous route. In someembodiments, the administration is via intratumoral route. In someembodiments, the individual is human.

In some embodiments, according to any of the methods described above,the method further comprising administering antigen presenting cells, orAPCs, (such as monocytes or monocyte-differentiated dendritic cells) tothe individual. Dendritic cells can be generated ex vivo via culturingmonocytes with specific cytokines (Palucka and Banchereau, NatureReviews Cancer 12: 265-277, 2012). In some embodiments, the APCs areadministered simultaneously with the effector cell composition. In someembodiments, the APCs are administered concurrently with the effectorcell composition. In some embodiments, the APCs are administeredsequentially with the effector cell composition. In some embodiments,the APCs are administered via the same route as the effector cellcomposition. In some embodiments, the APCs are administered to the samesite as the effector cell composition. In some embodiments, the effectorcell composition comprises the APCs.

Cancers

The anti-AMC constructs and anti-AMC CAR cells in some embodiments canbe useful for treating cancer. Cancers that may be treated using any ofthe methods described herein include tumors that are not vascularized,or not yet substantially vascularized, as well as vascularized tumors.The cancers may comprise non-solid tumors (such as hematological tumors,for example, leukemias and lymphomas) or may comprise solid tumors.Types of cancers to be treated with the anti-AMC constructs and anti-AMCCAR cells of the invention include, but are not limited to, carcinoma,blastoma, and sarcoma, and certain leukemia or lymphoid malignancies,benign and malignant tumors, and malignancies e.g., sarcomas,carcinomas, and melanomas. Adult tumors/cancers and pediatrictumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In some embodiments, the cancer is HCC. In some embodiments, the HCC isearly stage HCC, non-metastatic HCC, primary HCC, advanced HCC, locallyadvanced HCC, metastatic HCC, HCC in remission, or recurrent HCC. Insome embodiments, the HCC is localized resectable (i.e., tumors that areconfined to a portion of the liver that allows for complete surgicalremoval), localized unresectable (i.e., the localized tumors may beunresectable because crucial blood vessel structures are involved orbecause the liver is impaired), or unresectable (i.e., the tumorsinvolve all lobes of the liver and/or has spread to involve other organs(e.g., lung, lymph nodes, bone). In some embodiments, the HCC is,according to TNM classifications, a stage I tumor (single tumor withoutvascular invasion), a stage II tumor (single tumor with vascularinvasion, or multiple tumors, none greater than 5 cm), a stage III tumor(multiple tumors, any greater than 5 cm, or tumors involving majorbranch of portal or hepatic veins), a stage IV tumor (tumors with directinvasion of adjacent organs other than the gallbladder, or perforationof visceral peritoneum), N1 tumor (regional lymph node metastasis), orM1 tumor (distant metastasis). In some embodiments, the HCC is,according to AJCC (American Joint Commission on Cancer) stagingcriteria, stage T1, T2, T3, or T4 HCC. In some embodiments, the HCC isany one of liver cell carcinomas, fibrolamellar variants of HCC, andmixed hepatocellular cholangiocarcinomas.

In some embodiments, the cancer is a germ cell tumor.

Cancer treatments can be evaluated by, e.g., tumor regression, tumorweight or size shrinkage, time to progression, duration of survival,progression free survival, overall response rate, duration of response,quality of life, protein expression and/or activity. Approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of response through radiological imaging.

Methods for Diagnosis and Imaging Using Anti-AMC Constructs

Labeled anti-AMC antibody moieties and derivatives and analogs thereof,which specifically bind to an AMC on the surface of a cell, can be usedfor diagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the expression, aberrant expression and/oractivity of AFP, including any of the diseases and disorders describedabove, such as cancer (e.g., hepatocellular carcinoma, germ cell tumor,or breast cancer). For example, the anti-AMC antibody moieties of theinvention can be used in in situ, in vivo, ex vivo, and in vitrodiagnostic assays or imaging assays.

Additional embodiments of the invention include methods of diagnosing adisease or disorder associated with expression or aberrant expression ofAFP in an individual (e.g., a mammal, such as a human). The methodscomprise detecting AMC-presenting cells in the individual. In someembodiments, there is provided a method of diagnosing a disease ordisorder associated with expression or aberrant expression of AFP in anindividual (e.g., a mammal, such as a human) comprising (a)administering an effective amount of a labeled anti-AMC antibody moietyaccording to any of the embodiments described above to the individual;and (b) determining the level of the label in the individual, such thata level of the label above a threshold level indicates that theindividual has the disease or disorder. The threshold level can bedetermined by various methods, including, for example, by detecting thelabel according to the method of diagnosing described above in a firstset of individuals that have the disease or disorder and a second set ofindividuals that do not have the disease or disorder, and setting thethreshold to a level that allows for discrimination between the firstand second sets. In some embodiments, the threshold level is zero, andthe method comprises determining the presence or absence of the label inthe individual. In some embodiments, the method further compriseswaiting for a time interval following the administering of step (a) topermit the labeled anti-AMC antibody moiety to preferentiallyconcentrate at sites in the individual where the AMC is expressed (andfor unbound labeled anti-AMC antibody moiety to be cleared). In someembodiments, the method further comprises subtracting a background levelof the label. Background level can be determined by various methods,including, for example, by detecting the label in the individual priorto administration of the labeled anti-AMC antibody moiety, or bydetecting the label according to the method of diagnosing describedabove in an individual that does not have the disease or disorder. Insome embodiments, the disease or disorder is cancer. In someembodiments, the cancer is selected, for example, from the groupconsisting of hepatocellular carcinoma, germ cell tumor, and breastcancer. In some embodiments, the cancer is hepatocellular carcinoma. Insome embodiments, the cancer is metastatic hepatocellular carcinoma. Insome embodiments, the cancer is metastatic hepatocellular carcinoma, andthe method further comprises determining the level of the label in theindividual's blood. In some embodiments, the individual is human.

In some embodiments, there is provided a method of diagnosing metastatichepatocellular carcinoma in an individual (e.g., a mammal, such as ahuman), comprising (a) administering an effective amount of a labeledanti-AMC antibody moiety according to any of the embodiments describedabove to the individual; and (b) determining the level of the label inthe individual's blood, such that a level of the label above a thresholdlevel indicates that the individual has metastatic hepatocellularcarcinoma. The threshold level can be determined by various methods,including, for example, by detecting the label according to the methodof diagnosing described above in a first set of individuals that havemetastatic hepatocellular carcinoma and a second set of individuals thatdo not have metastatic hepatocellular carcinoma, and setting thethreshold to a level that allows for discrimination between the firstand second sets. In some embodiments, the threshold level is zero, andthe method comprises determining the presence or absence of the label inthe individual's blood. In some embodiments, the method furthercomprises waiting for a time interval following the administering ofstep (a) to permit the labeled anti-AMC antibody moiety topreferentially concentrate at sites in the individual where the AMC isexpressed (and for unbound labeled anti-AMC antibody moiety to becleared). In some embodiments, the method further comprises subtractinga background level of the label. Background level can be determined byvarious methods, including, for example, by detecting the label in theindividual prior to administration of the labeled anti-AMC antibodymoiety, or by detecting the label according to the method of diagnosingdescribed above in an individual that does not have metastatichepatocellular carcinoma. In some embodiments, the individual is human.

In some embodiments, there is provided a method of diagnosing a diseaseor disorder associated with expression or aberrant expression of AFP inan individual (e.g., a mammal, such as a human), comprising (a)contacting a labeled anti-AMC antibody moiety according to any of theembodiments described above with a sample (such as whole blood orhomogenized tissue) derived from the individual; and (b) determining thenumber of cells bound with the labeled anti-AMC antibody moiety in thesample, such that a value for the number of cells bound with the labeledanti-AMC antibody moiety above a threshold level indicates that theindividual has the disease or disorder. The threshold level can bedetermined by various methods, including, for example, by determiningthe number of cells bound with the labeled anti-AMC antibody moietyaccording to the method of diagnosing described above in a first set ofindividuals that have the disease or disorder and a second set ofindividuals that do not have the disease or disorder, and setting thethreshold to a level that allows for discrimination between the firstand second sets. In some embodiments, the threshold level is zero, andthe method comprises determining the presence or absence of cells boundwith the labeled anti-AMC antibody moiety in the sample. In someembodiments, the method further comprises subtracting a background levelof the number of cells bound with the labeled anti-AMC antibody moiety.Background level can be determined by various methods, including, forexample, by determining the number of cells bound with the labeledanti-AMC antibody moiety in the individual prior to administration ofthe labeled anti-AMC antibody moiety, or by determining the number ofcells bound with the labeled anti-AMC antibody moiety according to themethod of diagnosing described above in an individual that does not havethe disease or disorder. In some embodiments, the disease or disorder iscancer. In some embodiments, the cancer is selected, for example, fromthe group consisting of hepatocellular carcinoma, germ cell tumor, andbreast cancer. In some embodiments, the cancer is hepatocellularcarcinoma. In some embodiments, the cancer is metastatic hepatocellularcarcinoma. In some embodiments, the cancer is metastatic hepatocellularcarcinoma, and the sample is a blood sample (such as whole blood). Insome embodiments, the individual is human.

In some embodiments, there is provided a method of diagnosing metastatichepatocellular carcinoma in an individual (e.g., a mammal, such as ahuman), comprising (a) contacting a labeled anti-AMC antibody moietyaccording to any of the embodiments described above with a sample (suchas whole blood) derived from the individual; and (b) determining thenumber of cells bound with the labeled anti-AMC antibody moiety in thesample, such that a value for the number of cells bound with the labeledanti-AMC antibody moiety above a threshold level indicates that theindividual has metastatic hepatocellular carcinoma. The threshold levelcan be determined by various methods, including, for example, bydetermining the number of cells bound with the labeled anti-AMC antibodymoiety according to the method of diagnosing described above in a firstset of individuals that have metastatic hepatocellular carcinoma and asecond set of individuals that do not have metastatic hepatocellularcarcinoma, and setting the threshold to a level that allows fordiscrimination between the first and second sets. In some embodiments,the threshold level is zero, and the method comprises determining thepresence or absence of cells bound with the labeled anti-AMC antibodymoiety in the sample. In some embodiments, the method further comprisessubtracting a background level of the number of cells bound with thelabeled anti-AMC antibody moiety. Background level can be determined byvarious methods, including, for example, by determining the number ofcells bound with the labeled anti-AMC antibody moiety in the individualprior to administration of the labeled anti-AMC antibody moiety, or bydetermining the number of cells bound with the labeled anti-AMC antibodymoiety according to the method of diagnosing described above in anindividual that does not have metastatic hepatocellular carcinoma. Insome embodiments, the sample is blood (such as whole blood). In someembodiments, the individual is human.

Anti-AMC antibody moieties of the invention can be used to assay levelsof AMC-presenting cell in a biological sample using methods known tothose of skill in the art. Suitable antibody labels are known in the artand include enzyme labels, such as, glucose oxidase; radioisotopes, suchas iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), technetium (⁹⁹Tc,^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), samarium (¹⁵³Sm),lutetium (¹⁷⁷Lu), gadolinium (¹⁵⁹Gd), promethium (¹⁴⁹Pm), lanthanum(¹⁴⁰La), ytterbium (¹⁷⁵Yb), holmium (¹⁶⁶Ho), yttrium (⁹⁰Y), scandium(⁴⁷Sc), rhenium (¹⁸⁶Re, ¹⁸⁸Ro, ¹⁸⁸Re), praseodymium (¹⁴²Pr), rhodium(¹⁰⁵Rh), and ruthenium (⁹⁷Ru); luminol; fluorescent labels, such asfluorescein and rhodamine; and biotin.

Techniques known in the art may be applied to labeled anti-AMC antibodymoieties of the invention. Such techniques include, but are not limitedto, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos.5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425;5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003).Aside from the above assays, various in vivo and ex vivo assays areavailable to the skilled practitioner. For example, one can expose cellswithin the body of the subject to an anti-AMC antibody moiety which isoptionally labeled with a detectable label, e.g., a radioactive isotope,and binding of the anti-AMC antibody moiety to the cells can beevaluated, e.g., by external scanning for radioactivity or by analyzinga sample (e.g., a biopsy or other biological sample) derived from asubject previously exposed to the anti-AMC antibody moiety.

Articles of Manufacture and Kits

In some embodiments of the invention, there is provided an article ofmanufacture containing materials useful for the treatment of anAFP-positive disease such as cancer (for example hepatocellularcarcinoma, germ cell tumor, or breast cancer), for delivering ananti-AMC construct to a cell presenting an AMC on its surface, or forisolation or detection of AMC-presenting cells in an individual. Thearticle of manufacture can comprise a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. Generally, thecontainer holds a composition which is effective for treating a diseaseor disorder described herein, and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-AMC construct of theinvention. The label or package insert indicates that the composition isused for treating the particular condition. The label or package insertwill further comprise instructions for administering the anti-AMCconstruct composition to the patient. Articles of manufacture and kitscomprising combinatorial therapies described herein are alsocontemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In someembodiments, the package insert indicates that the composition is usedfor treating cancer (such as hepatocellular carcinoma, germ cell tumor,or breast cancer).

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., fortreatment of an AFP-positive disease or disorder described herein, fordelivering an anti-AMC construct to a cell presenting an AMC on itssurface, or for isolation or detection of AMC-presenting cells in anindividual, optionally in combination with the articles of manufacture.Kits of the invention include one or more containers comprising ananti-AMC construct composition (or unit dosage form and/or article ofmanufacture), and in some embodiments, further comprise another agent(such as the agents described herein) and/or instructions for use inaccordance with any of the methods described herein. The kit may furthercomprise a description of selection of individuals suitable fortreatment. Instructions supplied in the kits of the invention aretypically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

For example, in some embodiments, the kit comprises a compositioncomprising an anti-AMC construct (e.g., a full-length anti-AMC antibody,a multi-specific anti-AMC molecule (such as a bispecific anti-AMCantibody), or an anti-AMC immunoconjugate). In some embodiments, the kitcomprises a) a composition comprising an anti-AMC construct, and b) aneffective amount of at least one other agent, wherein the other agentincreases the expression of MHC class I proteins and/or enhances thesurface presentation of AFP peptides by MHC class I proteins (e.g.,IFNγ, IFNα, or Hsp90 inhibitor). In some embodiments, the kit comprisesa) a composition comprising an anti-AMC construct, and b) instructionsfor administering the anti-AMC construct composition to an individualfor treatment of an AFP-positive disease, such as HCC. In someembodiments, the kit comprises a) a composition comprising an anti-AMCconstruct, b) an effective amount of at least one other agent, whereinthe other agent increases the expression of MHC class I proteins and/orenhances the surface presentation of AFP peptides by MHC class Iproteins (e.g., IFNγ, IFNα, or Hsp90 inhibitor), and c) instructions foradministering the anti-AMC construct composition and the other agent(s)to an individual for treatment of an AFP-positive disease, such as HCC.The anti-AMC construct and the other agent(s) can be present in separatecontainers or in a single container. For example, the kit may compriseone distinct composition or two or more compositions wherein onecomposition comprises an anti-AMC construct and another compositioncomprises another agent.

In some embodiments, the kit comprises a) a composition comprising ananti-AMC construct (e.g., a full-length anti-AMC antibody, amulti-specific anti-AMC molecule (such as a bispecific anti-AMCantibody), or an anti-AMC immunoconjugate), and b) instructions forcombining the anti-AMC construct with cells (such as cells, e.g., immunecells, derived from an individual) to form a composition comprisinganti-AMC construct/cell conjugates and administering the anti-AMCconstruct/cell conjugate composition to the individual for treatment ofan AFP-positive disease (such as HCC). In some embodiments, the kitcomprises a) a composition comprising an anti-AMC construct, and b) acell (such as a cytotoxic cell). In some embodiments, the kit comprisesa) a composition comprising an anti-AMC construct, b) a cell (such as acytotoxic cell), and c) instructions for combining the anti-AMCconstruct with the cell to form a composition comprising anti-AMCconstruct/cell conjugates and administering the anti-AMC construct/cellconjugate composition to an individual for the treatment of anAFP-positive disease, such as HCC. In some embodiments, the kitcomprises a composition comprising an anti-AMC construct in associationwith a cell (such as an cytotoxic cell). In some embodiments, the kitcomprises a) a composition comprising an anti-AMC construct inassociation with a cell (such as a cytotoxic cell), and b) instructionsfor administering the composition to an individual for the treatment ofan AFP-positive disease, such as HCC. In some embodiments, theassociation is by conjugation of the anti-AMC construct to a molecule onthe surface of the cell. In some embodiments, the association is byinsertion of a portion of the anti-AMC construct into the outer membraneof the cell.

In some embodiments, the kit comprises a nucleic acid (or set of nucleicacids) encoding an anti-AMC construct (e.g., a full-length anti-AMCantibody, a multi-specific anti-AMC molecule (such as a bispecificanti-AMC antibody), an anti-AMC CAR, or an anti-AMC immunoconjugate) orpolypeptide portions thereof. In some embodiments, the kit comprises a)a nucleic acid (or set of nucleic acids) encoding an anti-AMC constructor polypeptide portions thereof, and b) a host cell (such as an effectorcell) for expressing the nucleic acid (or set of nucleic acids). In someembodiments, the kit comprises a) a nucleic acid (or set of nucleicacids) encoding an anti-AMC construct or polypeptide portions thereof,and b) instructions for i) expressing the anti-AMC construct in a hostcell (such as an effector cell, e.g., a T cell), ii) preparing acomposition comprising the anti-AMC construct or the host cellexpressing the anti-AMC construct, and iii) administering thecomposition comprising the anti-AMC construct or the host cellexpressing the anti-AMC construct to an individual for the treatment ofan AFP-positive disease, such as HCC. In some embodiments, the host cellis derived from the individual. In some embodiments, the kit comprisesa) a nucleic acid (or set of nucleic acids) encoding an anti-AMCconstruct or polypeptide portions thereof, b) a host cell (such as aneffector cell) for expressing the nucleic acid (or set of nucleicacids), and c) instructions for i) expressing the anti-AMC construct inthe host cell, ii) preparing a composition comprising the anti-AMCconstruct or the host cell expressing the anti-AMC construct, and iii)administering the composition comprising the anti-AMC construct or thehost cell expressing the anti-AMC construct to an individual for thetreatment of an AFP-positive disease, such as HCC.

In some embodiments, the kit comprises a nucleic acid encoding ananti-AMC CAR. In some embodiments, the kit comprises a vector comprisinga nucleic acid encoding an anti-AMC CAR. In some embodiments, the kitcomprises a) a vector comprising a nucleic acid encoding an anti-AMCCAR, and b) instructions for i) introducing the vector into effectorcells, such as T cells derived from an individual, ii) preparing acomposition comprising the anti-AMC CAR effector cells, and iii)administering the anti-AMC CAR effector cell composition to theindividual for treatment of an AFP-positive disease, such as HCC.

The kits of the invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Kits mayoptionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The instructions relating to the use of the anti-AMC constructcompositions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. For example, kits may be provided that containsufficient dosages of an anti-AMC construct (e.g., a full-lengthanti-AMC antibody, a multi-specific anti-AMC molecule (such as abispecific anti-AMC antibody), an anti-AMC CAR, or an anti-AMCimmunoconjugate) as disclosed herein to provide effective treatment ofan individual for an extended period, such as any of a week, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9months, or more. Kits may also include multiple unit doses of theanti-AMC construct and pharmaceutical compositions and instructions foruse and packaged in quantities sufficient for storage and use inpharmacies, for example, hospital pharmacies and compounding pharmacies.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

Exemplary Embodiments

Embodiment 1. In some embodiments, there is provided an isolatedanti-AMC construct comprising an antibody moiety that specifically bindsto a complex comprising an alpha-fetoprotein (AFP) peptide and a majorhistocompatibility (MHC) class I protein (an AFP/MHC class I complex, orAMC).

Embodiment 2. In some further embodiments of embodiment 1, the AFP/MHCclass I complex is present on a cell surface.

Embodiment 3. In some further embodiments of embodiment 1, the AFP/MHCclass I complex is present on the surface of a cancer cell.

Embodiment 4. In some further embodiments of any one of embodiments 1-3,the MHC class I protein is human leukocyte antigen (HLA)-A.

Embodiment 5. In some further embodiments of embodiment 4, the MHC classI protein is HLA-A02.

Embodiment 6. In some further embodiments of embodiment 5, the MHC classI protein is the HLA-A*02:01 subtype of the HLA-A02 allele.

Embodiment 7. In some further embodiments of any one of embodiments 1-6,the antibody moiety cross-reacts with a complex comprising the AFPpeptide and a second MHC class I protein having a different HLA allelethan the MHC class I protein.

Embodiment 8. In some further embodiments of any one of embodiments 1-7,the AFP peptide is 8 to 12 amino acids in length.

Embodiment 9. In some further embodiments of any one of embodiments 1-8,the AFP peptide is derived from human AFP.

Embodiment 10. In some further embodiments of any one of embodiments1-9, the AFP peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3-13 and 16.

Embodiment 11. In some further embodiments of embodiment 9, the AFPpeptide has the amino acid sequence of FMNKFIYEI (SEQ ID NO: 4).

Embodiment 12. In some further embodiments of any one of embodiments1-11, the isolated anti-AMC construct cross-reacts with a complexcomprising an interspecies variant of the AFP peptide and the MHC classI protein.

Embodiment 13. In some further embodiments of any one of embodiments1-12, the antibody moiety is human, humanized, or semi-synthetic.

Embodiment 14. In some further embodiments of any one of embodiments1-13, the antibody moiety is a full-length antibody, a Fab, a Fab′, a(Fab′)2, an Fv, or a single chain Fv (scFv).

Embodiment 15. In some further embodiments of any one of embodiments1-14, the antibody moiety binds to the AFP/MHC class I complex with anequilibrium dissociation constant (Kd) from about 0.1 pM to about 500nM.

Embodiment 16. In some further embodiments of any one of embodiments1-15, the isolated anti-AMC construct binds to the AFP/MHC class Icomplex with a Kd from about 0.1 pM to about 500 nM.

Embodiment 17. In some further embodiments of any one of embodiments1-16, the antibody moiety comprises:

i) a heavy chain variable domain comprising a heavy chaincomplementarity determining region (HC-CDR) 1 comprising the amino acidsequence of G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/W (SEQ ID NO: 87), or avariant thereof comprising up to about 3 amino acid substitutions, anHC-CDR2 comprising the amino acid sequence of I/S-K/S-X-H/Y-X-G-X-T (SEQID NO: 88), or a variant thereof comprising up to about 3 amino acidsubstitutions, and an HC-CDR3 comprising the amino acid sequence ofA/G-X-W/Y-Y-X-X-X-F/Y-D (SEQ ID NO: 89); or a variant thereof comprisingup to about 3 amino acid substitutions; and

ii) a light chain variable domain comprising a light chaincomplementarity determining region (LC-CDR) 1 comprising the amino acidsequence of S/T-G/S-D/N-IN-A/G-A/SN-X-H/Y (SEQ ID NO: 120), or a variantthereof comprising up to about 3 amino acid substitutions, and anLC-CDR3 comprising the amino acid sequence of Q-S/T-Y/W-D/T-S/T-A/S (SEQID NO: 121); or a variant thereof comprising up to 3 amino acidsubstitutions, wherein

X can be any amino acid.

Embodiment 18. In some further embodiments of any one of embodiments1-16, the antibody moiety comprises:

i) a heavy chain variable domain comprising an HC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 57-66, or a variantthereof comprising up to about 5 amino acid substitutions, an HC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 67-76, or avariant thereof comprising up to about 5 amino acid substitutions, andan HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; or a variant thereof comprising up to about 5 amino acidsubstitutions; and

ii) a light chain variable domain comprising an LC-CDR1 comprising theamino acid sequence of any one of SEQ ID NOs: 90-99, or a variantthereof comprising up to about 5 amino acid substitutions, an LC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 100-109, ora variant thereof comprising up to about 3 amino acid substitutions, andan LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:110-119; or a variant thereof comprising up to about 5 amino acidsubstitutions.

Embodiment 19. In some further embodiments of any one of embodiments1-16, the antibody moiety comprises:

i) a heavy chain (HC) variable domain comprising an HC-CDR1 comprisingthe amino acid sequence of any one of SEQ ID NOs: 57-66, an HC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 67-76, andan HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs:77-86; or a variant thereof comprising up to about 5 amino acidsubstitutions in the HC-CDR regions; and

ii) a light chain (LC) variable domain comprising an LC-CDR1 comprisingthe amino acid sequence of any one of SEQ ID NOs: 90-99, an LC-CDR2comprising the amino acid sequence of any one of SEQ ID NOs: 100-109,and an LC-CDR3 comprising the amino acid sequence of any one of SEQ IDNOs: 110-119; or a variant thereof comprising up to about 5 amino acidsubstitutions in the LC-CDR regions.

Embodiment 20. In some further embodiments of embodiment 18 or 19, theantibody moiety comprises a) a heavy chain variable domain comprisingthe amino acid sequence of any one of SEQ ID NOs: 17-26, or a variantthereof having at least about 95% sequence identify to any one of SEQ IDNOs: 17-26; and b) a light chain variable domain comprising the aminoacid sequence of any one of SEQ ID NOs: 27-36, or a variant thereofhaving at least about 95% sequence identity to any one of SEQ ID NOs:27-36.

Embodiment 21. In some further embodiments of embodiment 20, theantibody moiety comprises a heavy chain variable domain comprising theamino acid sequence of any one of SEQ ID NOs: 17-26 and a light chainvariable domain comprising the amino acid sequence of any one of SEQ IDNOs: 27-36.

Embodiment 22. In some further embodiments of any one of embodiments1-21, the isolated anti-AMC construct is a full-length antibody.

Embodiment 23. In some further embodiments of any one of embodiments1-22, the isolated anti-AMC construct is monospecific.

Embodiment 24. In some further embodiments of any one of embodiments1-22, the isolated anti-AMC construct is multispecific.

Embodiment 25. In some further embodiments of embodiment 24, theisolated anti-AMC construct is bispecific.

Embodiment 26. In some further embodiments of embodiment 24 or 25, theisolated anti-AMC construct is a tandem scFv, a diabody (Db), a singlechain diabody (scDb), a dual-affinity retargeting (DART) antibody, adual variable domain (DVD) antibody, a knob-into-hole (KiH) antibody, adock and lock (DNL) antibody, a chemically cross-linked antibody, aheteromultimeric antibody, or a heteroconjugate antibody.

Embodiment 27. In some further embodiments of embodiment 26, theisolated anti-AMC construct is a tandem scFv comprising two scFvs linkedby a peptide linker.

Embodiment 28. In some further embodiments of embodiment 27, the peptidelinker comprises the amino acid sequence GGGGS.

Embodiment 29. In some further embodiments of any one of embodiments24-28, the isolated anti-AMC construct further comprises a secondantibody moiety that specifically binds to a second antigen.

Embodiment 30. In some further embodiments of embodiment 29, the secondantigen is an antigen on the surface of a T cell.

Embodiment 31. In some further embodiments of embodiment 30, the secondantigen is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3,CD28, OX40, GITR, CD137, CD27, CD40L, and HVEM.

Embodiment 32. In some further embodiments of embodiment 30, the secondantigen is CD3ε, and the isolated anti-AMC construct is a tandem scFvcomprising an N-terminal scFv specific for the AFP/MHC class I complexand a C-terminal scFv specific for CD3ε.

Embodiment 33. In some further embodiments of embodiment 30, the T cellis selected from the group consisting of a cytotoxic T cell, a helper Tcell, and a natural killer T cell.

Embodiment 34. In some further embodiments of embodiment 29, the secondantigen is an antigen on the surface of a natural killer cell, aneutrophil, a monocyte, a macrophage or a dendritic cell.

Embodiment 35. In some further embodiments of any one of embodiments1-21, the isolated anti-AMC construct is a chimeric antigen receptor.

Embodiment 36. In some further embodiments of embodiment 35, thechimeric antigen receptor comprises an extracellular domain comprisingthe antibody moiety, a transmembrane domain, and an intracellularsignaling domain comprising a CD3ζ intracellular signaling sequence anda CD28 intracellular signaling sequence.

Embodiment 37. In some further embodiments of any one of embodiments1-21, the isolated anti-AMC construct is an immunoconjugate comprisingthe antibody moiety and an effector molecule.

Embodiment 38. In some further embodiments of embodiment 36, theeffector molecule is a therapeutic agent selected from the groupconsisting of a drug, a toxin, a radioisotope, a protein, a peptide, anda nucleic acid.

Embodiment 39. In some further embodiments of embodiment 38, thetherapeutic agent is a drug or a toxin.

Embodiment 40. In some further embodiments of embodiment 37, theeffector molecule is a label.

Embodiment 41. In some embodiments there is provided a pharmaceuticalcomposition comprising the isolated anti-AMC construct of any one ofembodiments 1-39.

Embodiment 42. In some embodiments there is provided a host cellexpressing the isolated anti-AMC construct of any one of embodiments1-40.

Embodiment 43. In some embodiments there is provided a nucleic acidencoding the polypeptide components of the isolated anti-AMC constructof any one of embodiments 1-40.

Embodiment 44. In some embodiments there is provided a vector comprisingthe nucleic acid of embodiment 43.

Embodiment 45. In some embodiments there is provided an effector cellexpressing the isolated anti-AMC construct of embodiment 35 or 36.

Embodiment 46. In some further embodiments of embodiment 45, theeffector cell is a T cell.

Embodiment 47. In some embodiments there is provided a method ofdetecting a cell presenting a complex comprising an AFP peptide and anMHC class I protein on its surface, comprising contacting the cell withthe isolated anti-AMC construct of embodiment 40 and detecting thepresence of the label on the cell.

Embodiment 48. In some embodiments there is provided a method oftreating an individual having an AFP-positive disease, comprisingadministering to the individual an effective amount of thepharmaceutical composition of embodiment 41.

Embodiment 49. In some embodiments there is provided a method oftreating an individual having an AFP-positive disease, comprisingadministering to the individual an effective amount of the effector cellof embodiments 45 or 46.

Embodiment 50. In some further embodiments of embodiment 48 or 49, theadministration is via intravenous route.

Embodiment 51. In some further embodiments of embodiment 48 or 49, theadministration is via intratumoral route.

Embodiment 52. In some further embodiments of embodiment 48 or 49, theadministration is to an injection site distal to a first disease site.

Embodiment 53. In some further embodiments of embodiment 52, theinjection site is a first tumor distal to the first disease site.

Embodiment 54. In some further embodiments of embodiment 52 or 53, thefirst disease site is an AFP-positive tumor.

Embodiment 55. In some embodiments there is provided a method ofdiagnosing an individual having an AFP-positive disease, comprising:

a) administering an effective amount of the isolated anti-AMC constructof embodiment 40 to the individual; and

b) determining the level of the label in the individual, wherein a levelof the label above a threshold level indicates that the individual hasthe AFP-positive disease.

Embodiment 56. In some embodiments there is provided a method ofdiagnosing an individual having an AFP-positive disease, comprising:

a) contacting a sample derived from the individual with the isolatedanti-AMC construct of embodiment 40; and

b) determining the number of cells bound with the isolated anti-AMCconstruct in the sample, wherein a value for the number of cells boundwith the isolated anti-AMC construct above a threshold level indicatesthat the individual has the AFP-positive disease.

Embodiment 57. In some further embodiments of any one of embodiments48-56, the AFP-positive disease is cancer.

Embodiment 58. In some further embodiments of embodiment 57, the canceris hepatocellular carcinoma, germ cell tumor, or breast cancer.

Embodiment 59. In some further embodiments of embodiment 58, the canceris hepatocellular carcinoma.

Embodiment 60. In some further embodiments of embodiment 59, the canceris metastatic hepatocellular carcinoma.

Embodiment 61. In some further embodiments of any one of embodiments48-54, the AFP-positive disease is hepatocellular carcinoma andmetastasis is inhibited.

EXAMPLES Materials Cell Samples, Cell Lines, and Antibodies

The cell lines HepG2, SK-Hep1, MCF7, Malme-3M, CA46, THP-1, Colo205,ASPC1, OVCAR3, LnCAP, A498 and Hela were obtained from the American TypeCulture Collection. SK-Hep1-MiniG (or SK-Hep1 AFP MG), MCF7-MiniG andHela-MiniG (or Hela-MG) were generated by transducing the respectiveparental cell lines with an AFP158 peptide expressing minigene cassette,which results in a high level of cell surface expression ofAFP158/HLA-A*02:01 complex in SK-Hep1 and MCF7 cells. Hela is HLA-A03-and HLA-A68-positive and HLA*A02:01-negative, and therefore Hela-MiniGserved as AFP158 minigene expressing, but AFP158/HLA*A02:01 negativecontrol cell line. The cell lines were cultured in RPMI 1640supplemented with 10% FBS, 2 mM glutamine at 37° C./5% CO₂.

The following antibodies were purchased: monoclonal Ab against humanHLA-A02 (clone BB7.2) conjugated to FITC or APC, and its isotype controlmouse IgG2b/FITC or APC to human or mouse CD3, CD 19, CD56, CD33, CD34(BD Biosciences, San Diego), goat F(ab)₂ anti-human IgG conjugated to PEor FITC and goat F(ab)₂ anti-mouse IgG (Invitrogen).

All peptides were synthesized by Genemed Synthesis, Inc. (San Antonio,Tex.). Peptides were >90% pure. The peptides were dissolved in DMSO anddiluted in saline at 5 mg/mL and frozen at −180° C. Biotinylated singlechain AFP peptide/HLA-A*02:01 and control peptide/HLA-A*02:01 complexeswere synthesized by refolding the peptides with recombinant HLA-A*02:01and β-2 microglobulin (β2M). 19 control peptides (SEQ ID NOs: 122-140)that bind HLA-A*02:01 were generated from the following 15 genes: BCR,BTG2, CALR, CD247, CSF2RA, CTSG, DDXS, DMTN, HLA-E, IFI30, IL7, PIM1,PPP2R1B, RPS6KB1, and SSR1.

Example 1. Production of Biotinylated AFP158/HLA-A*02:01 Complex Monomer

Biotinylated AFP158/HLA-A*02:01 complex monomers were prepared accordingto standard protocols (Altman, J. D. & Davis, M. M., Current Protocolsin Immunology 17.3.1-17.3.33, 2003). In brief, DNA encoding full-lengthhuman β-2 microglobulin (β2M) was synthesized by Genewiz and cloned intovector pET-27b. The BirA substrate peptide (BSP) was added to theC-terminus of HLA-A*02:01 extracellular domain (ECD). DNA encodingHLA-A*02:01 ECD-BSP was also synthesized by Genewiz and cloned intovector pET-27b. The vectors expressing human 132M and HLA-A*02:01ECD-BSP were transformed into E. coli BL21 cells separately, andisolated as inclusion bodies from bacterial culture. Peptide ligandAFP158 refolded with human 132M and HLA-A*02:01 ECD-BSP to formAFP158/HLA-A*02:01 complex monomer. Folded peptide/HLA-A*02:01 monomerswere concentrated by ultrafiltration and further purified throughsize-exclusion chromatography. HiPrep 26/60 Sephacryl S-300 HR wasequilibrated with 1.5 column volumes of Hyclone Dulbecco's PhosphateBuffered Saline solution (Thermo Scientific, Cat No. SH3002802). Theunpurified sample was loaded and eluted with 1 column volume. The firstpeak, corresponding to misfolded aggregates, eluted at approximately111.3 mL, the peak corresponding to the properly folded MHC complex wasobserved at 212.5 mL, and the peak corresponding to free 132M wasobserved at 267.2 mL (FIG. 1). SDS-PAGE of the purified AFP158/MHCcomplex was performed to determine protein purity. In brief, 1 μg of theprotein complex was mixed with 2.54 of the NuPAGE LDS Sample Buffer(Life Technologies, NP0008) and brought up to 104 with deionized water.The sample was heated at 70° C. for 10 minutes, and then loaded onto thegel. Gel electrophoresis was performed at 180V for 1 hour. HLA-A*02:01and β2M subunits were observed as the major bands on the gel (FIG. 2).Peptide/HLA-A*02:01 monomers were biotinylated via BirA-mediatedenzymatic reaction and subsequently purified by high-resolutionanion-exchange chromatography. Biotinylated peptide/HLA-A*02:01 monomerswere stored in PBS at −80° C.

Example 2. Selection and Characterization of scFv Specific forAFP158/HLA-A*02:01 Complexes

A collection of human scFv antibody phage display libraries(diversity=10×10¹⁰) constructed by Eureka Therapeutics was used for theselection of human mAbs specific to AFP158/HLA-A*02:01. 15 fully humanphage scFv libraries were used to pan against AFP158/HLA-A*02:01complex. In order to reduce the conformational change of MHC1 complexintroduced by immobilizing the protein complex onto plastic surfaces,solution panning and cell panning were used in place of conventionalplate panning. In solution panning, biotinylated antigens were firstmixed with the human scFv phage library after extended washing with PBSbuffer, and then antigen-scFv antibody phage complexes were pulled downby streptavidin-conjugated Dynabeads M-280 through a magnetic rack. Thebound clones were then eluted and used to infect E. coli XL1-Blue cells.In cell panning, T2 cells loaded with AFP158 peptide were first mixedwith the human scFv phage library. T2 cells are a TAP-deficient,HLA-A*02:01⁺ lymphoblast cell line. To load peptide, T2 cells werepulsed with peptides (50 ug/ml) in serum-free RPMI1640 medium in thepresence of 20 μg/ml β2M overnight. After extended washing with PBS,peptide-loaded T2 cells with bound scFv antibody phage were spun down.The bound clones were then eluted and used to infect E. coli XL1-Bluecells. The phage clones expressed in bacteria were then purified. Thepanning was performed for 3-4 rounds with either solution panning, cellpanning or a combination of solution and cell panning to enrich for scFvphage clones that bound AFP158/HLA-A*02:01 specifically.

Streptavidin ELISA plates were coated with biotinylatedAFP158/HLA-A*02:01 complex monomer (Bio-AFP158) or biotinylated controlpeptide/HLA-A*02:01 monomer (Bio-control), respectively. Individualphage clones from enriched phage display panning pools againstAFP158/HLA-A*02:01 complex were incubated in the coated plates. Bindingof the phage clones was detected by HRP-conjugated anti-M13 antibodiesand developed using HRP substrate. The absorbance was read at 450 nm.605 positive clones were identified through ELISA screening of 1260phage clones enriched from phage panning. FIG. 3 provides an example ofphage clone binding to biotinylated AFP158/HLA-A*02:01 monomer in anELISA assay. 82 unique clones were identified by DNA sequencing of the605 ELISA positive phage clones. Positive clones were determined bystandard ELISA against biotinylated AFP158/HLA-A*02:01 complex monomer.Then, unique antibody clones were identified through DNA sequencing ofELISA-positive clones. Specific and unique clones were further testedfor their binding to HLA-A2/peptide complexes on live cell surfaces byflow cytometry (FACS analysis) using AFP158-loaded live T2 cells. T2cells loaded with different peptides and β2M were first stained withpurified scFv phage clones, followed by staining with a mouse anti-M13mAb, and finally the R-PE conjugated horse anti-mouse IgG from VectorLabs. Each step of the staining was done between 30-60 minutes on iceand the cells were washed twice between staining. Among the 82 clones,44 recognize AFP158-loaded T2 cells specifically. FIG. 4 provides anexample of AFP158/HLA-A*02:01 specific phage clone binding topeptide-loaded T2 cells through FACS. The phage clones specificallybound to AFP158-loaded T2 cells and did not recognize T2 cells loadedwith hTERT-derived peptide ILAKFLHWL (hTERT540, SEQ ID NO: 141) in thecontext of HLA-A*02:01, or T2 cells with β2M but without peptide loaded.

Example 3. Characterization of FACS-Positive AFP158-Specific PhageClones Cross-Reactivity to Mouse AFP158 Peptide

Clones selected from FACS binding analysis against AFP158-loaded T2cells were characterized further for cross-reactivity towards mouseAFP158 peptide/HLA-A*02:01 complex on live cell surfaces by FACSanalysis using mouse AFP158-loaded live T2 cells. Mouse AFP158 peptidediffers from human AFP158 peptide by two amino acids at position 4 andposition 9. The mouse AFP158 peptide sequence is FMNRFIYEV (SEQ ID NO:16), while the human AFP158 peptide sequence is FMNKFIYEI (SEQ ID NO:4). Antibodies cross-reacting with both human and mouse AFP158peptide/MHC complexes are useful for assessing antibody drug toxicity inHLA-A*02:01 transgenic mice. Among the phage clones tested, 4 clones(#17, #33, #48 and #76) recognized both human AFP158- and mouseAFP158-loaded T2 cells (FIG. 5). Clones #52 and #79 also bound to mouseAFP158-loaded T2 cells, but to a lesser degree.

Epitope Mapping by Alanine Walking

To investigate with precision the epitope for the mAb recognition, humanAFP158 peptides with alanine substitutions at positions 1, 3, 4, 5, 6, 7and 8 (SEQ ID NOs: 7-13) were pulsed onto T2 cells. Antibody phageclones were then tested for binding to these peptide-loaded T2 cells byFACS analysis. Although all the antibodies recognized the smallconformational epitope formed by the AFP158 peptide and its surroundingMHCα chain residues, the key peptide residues interacting with differentantibodies were quite different. For example, clone #52 is predicted tobind to the N-terminal half of the AFP158 peptide since alaninesubstitution at position 1 or 3 dramatically reduced binding to thepeptide-loaded T2 cells, and alanine substitution at position 4completely abrogated the binding of clone #52. In contrast, alaninesubstitutions at position 5, 6, 7 or 8 didn't change the binding ofclone #52. Clone #17-13, on the other hand, was insensitive to alaninesubstitution at position 4, but was sensitive to changes at position 3,5 and 7. FIG. 6 provides an example of FACS analysis, showing thebinding of phage clone #52 to T2 cells loaded with the various AFPpeptides. Table 6 summaries the binding of severalAFP158/HLA-A*02:01-specific antibody clones to alanine-substituted humanAFP158 peptide-loaded T2 cells.

TABLE 6 Peptide Ala Position ET1402-52 ET1402-61 ET1402-76 ET1402-79AM1402-17-13 FMNKFIYEI 37900 30400 14400 30100 83300 AMNKFIYEI 1 46812300 5458 21000 55600 FMAKFIYEI 3 4470 38600 38200 786 2914 FMNAFIYEI 420.9 15.1 38.3 79.2 50800 FMNKAIYEI 5 21800 3719 49300 34.4 1500FMNKFAYEI 6 18800 169 40300 27.7 28800 FMNKFIAEI 7 42500 1110 29300 907572 FMNKFIYAI 8 38500 1748 53300 16100 43200 Sensitive 4, 1, 3 4, 6, 7,8, 5 4, 1 5, 6, 4, 3, 7 7, 5, 6 Position

Antibody Binding Specificity Evaluation Against Endogenous Peptides

On average, each nucleated cell in the human body expresses about half amillion different peptide/MHC class I complexes. In order to developanti-peptide/MHCI-complex antibodies into anti-cancer drugs with highspecificity and therapeutic index, it is essential for the antibodies tospecifically recognize the target peptide/MHCI complex, but not the MHCImolecule itself, or MHCI molecules bound to other peptides presented oncell surfaces. For the current study, the relevant MHCI molecule isHLA-A*02:01. During the early stages of our phage panning and screening,we eliminated antibodies that bound to the HLA-A*02:01 molecule alone(see, for example, FIGS. 3 and 4). The top phage clones were alsoscreened against 19 endogenous HLA-A*02:01 peptides, which were derivedfrom proteins normally expressed in multiple types of nucleated humancells, such as globin a chain, 13 chain, nuclear protein p68, and thelike. The pool of endogenous peptides (P19, SEQ ID NOs: 122-140) wasloaded into T2 cells and antibody binding was determined through FACSanalysis. As shown in FIG. 7, the AFP158/HLA-A*02:01-specific antibodyphage clones bound AFP158 peptide-loaded T2 cells, but not T2 cellsloaded with endogenous peptides. We conclude that the identifiedantibodies are specific to AFP158 peptide/HLA-A*02:01 complexes, and donot recognize HLA-A*02:01 molecules bound to other HLA-A*02:01 peptidestested.

Example 4. Engineering Bispecific Antibodies

Bispecific antibodies (BsAbs) were generated using scFv sequences of theAFP158/HLA-A*02:01-specific phage clones. The BsAbs are single-chainbispecific antibodies comprising the scFv sequence of anAFP158/HLA-A*02:01-specific phage clone at the N-terminal end and ananti-human CD3ε mouse monoclonal scFv at the C-terminal end (Brischwein,K. et al., Mol. Immunol. 43:1129-1143, 2006). The DNA fragments codingfor the AFP158 scFv and the anti-human CD3ε scFv were synthesized byGenewiz and subcloned into Eureka's mammalian expression vector pGSN-Hygusing standard DNA technology. A hexhistamine tag was inserted at theC-terminal end for antibody purification and detection. Chinese hamsterovary (CHO) cells were transfected with the BsAb expression vector, andthen cultured for 7 days for BsAb antibody production. CHO cellsupernatants containing secreted AFP158 BsAb molecules were collected.BsAb antibodies were purified using HisTrap HP column (GE healthcare) byFPLC AKTA system. Briefly, CHO cell culture was clarified and loadedonto the column with low imidazole concentration (20 mM), and then anisocratic high imidazole concentration elution buffer (500 mM) was usedto elute the bound BsAb proteins. Molecular weights of the purifiedAFP158 BsAbs antibodies were measured under non-reducing conditions bygel electrophoresis. 4 μg of the protein was mixed with 2.54 of theNuPAGE LDS Sample Buffer (Life Technologies, NP0008) and brought up to10 μL with deionized water. The sample was heated at 70° C. for 10minutes, and then loaded onto the gel. Gel electrophoresis was performedat 180V for 1 hour. ˜50 KD bands are observed as the major bands on thegel (FIG. 8). Antibody aggregation was assessed by size-exclusionchromatography (SEC). 504 of the sample was injected into the SEC column(Agilent, BioSEC-3,300A, 4.6×300 mm) while flowing a buffer consistingof Dulbecco's Phosphate Buffered Saline (Fisher Scientific, SH30028.FS)and 0.2M arginine adjusted to pH 7.0. BsAb was observed as the majorpeak at the retention time ˜15.8 mL. (FIG. 9). BsAbs with high molecularweight aggregation less than 10% were used for further characterization.

Example 5. Characterization of AFP158 BsAb Antibodies Binding Affinityof AFP158 BsAb Antibodies

The binding affinity of AFP158 BsAb antibodies to recombinantAFP158/HLA-A*02:01 complex was measured by Surface Plasma Resonance(BiaCore). The binding parameters between the AFP158 BsAb and theAFP158/HLA-A*02:01 complex were measured using a His Capture Kit (GEHealthcare, Cat #28995056) on a Biacore X100 (GE Healthcare) accordingto the manufacturer's protocol for multi-cycle kinetics measurement. Allof the proteins used in the assay were diluted using HBS-E buffer. Inbrief, 1 μg/mL of the AFP158 BsAb was immobilized onto a Sensor Chippre-functionalized with the anti-histidine antibody by flowing thesolution through the flow cell 2 at 2 μL/min for 2 minutes. Bindingtowards the AFP158/A*02:01 complex was analyzed at 0.19, 0.38, 7.5, 15,and 30 μg/mL, each run consisting of a 3 minute association and 3 minutedissociation at 30 μL/min. At the end of cycle, the surface wasregenerated using the regeneration buffer from the His Capture kit.Following the kinetics measurement, the surface was regenerated usingthe regeneration solution from the kit. The data were analyzed using 1:1binding site mode with the BiaCore X-100 evaluation software. Thebinding parameters (association on rate constant k_(a), dissociationconstant k_(d), and equilibrium dissociation constant K_(d)) werecalculated. The binding affinities of AFP158 antibodies, in themonovalent scFv format, fall into the range of 10-500 nM. (Examplesprovided in Table 7).

TABLE 7 Clone # k_(a) [1/Ms] k_(d) [1/s] K_(d) [nM] #52 2.63E+05 1.71E−265 #61 8.92E+04 3.08E−3 34 #76 9.94E+04 1.42E−2 143 #79 7.88E+04 2.51E−2318 #17-13 7.36E+04 3.45E−3 47

T-Cell Killing Assay

Tumor cytotoxicity was assayed by LDH Cytotoxicity Assay (Promega).Human T cells purchased from AllCells were activated and expanded withCD3/CD28 Dynabeads (Invitrogen) according to manufacturer's protocol.Activated T cells (ATC) were cultured and maintained in RPMI1640 mediumwith 10% FBS plus 100 U/ml IL-2, and used at day 7-14. The T cellswere >99% CD3⁺ by FACS analysis. Activated T cells (Effector cells) andTarget cells were co-cultured at a 5:1 ratio with differentconcentrations of BsAb antibodies for 16 hours. Cytotoxicities were thendetermined by measuring LDH activities in culture supernatants.

AFP158 BsAb antibodies killed cancer cells in an AFP andHLA-A*02:01-dependent manner. Among all cell lines tested, HEPG2 (AFPand HLA-A*02:01 positive) was most effectively killed by T cellsredirected through AFP158 BsAbs. Other cell lines tested that wereeither AFP negative or HLA-A02 negative, or negative for both were notkilled as effectively under the same experimental settings (FIG. 10).

Cross-Reactivity of AFP158 BsAb Antibodies Against Multiple HLA-A02Alleles

Human MHCI molecules consist of 6 class isoforms, HLA-A, -B, -C, -E, -Fand G. The HLA-A, -B and -C heavy chain genes are highly polymorphic.For each isoform, the HLA genes are further grouped according to thesimilarity of heavy chain sequences. For example, HLA-A is divided intodifferent alleles such as HLA-A01, -A02, -A03, etc. For the HLA-A02allele, there are multiple subtypes, such as HLA-A*02:01, A*02:02, etc.Between the different subtypes of HLA-A02 group, the sequencedifferences are limited to only several amino acids. So in many cases,peptides that bind to HLA-A*02:01 molecule can also form complexes withmultiple subtypes of the HLA-A02 allele. As shown in Table 8 (world wideweb.allelefrequencies.net/), although HLA-A*02:01 is the dominantHLA-A02 subtype among Caucasian populations, in Asia and Africa,A*02:03, A*02:05, A*02:06, A*02:07 and A*02:11 are also common HLA-A02subtypes. The ability of AFP158 antibodies to recognize not only AFP158peptide in the context of HLA-A*02:01, but also other subtypes ofHLA-A02, will broaden the patient population that might be able tobenefit from AFP158 antibody drug treatment. We therefore generatedrecombinant AFP158/MHCI complexes with other subtypes of the HLA-A02allele and tested the binding affinity of theAFP158/HLA-A*02:01-specific antibodies for these other complexes.Binding affinity was determined using a ForteBio Octet QK. 5 μg/mLbiotinylated HLA-A02 MHC complex of varying subtypes was loaded onto astreptavidin biosensor. After washing off excess antigen, BsAbantibodies were tested at 10 μg/mL for association and dissociation.Binding parameters were calculated using a 1:1 binding site, partial fitmodel. Table 9 shows binding affinities of several AFP158 BsAbs formultiple AFP158/HLA-A02 complexes with different subtypes. All of theantibodies tested were found to recognize AFP158 bound to multiplesubtypes of the HLA-A02 allele.

TABLE 8 sub- north saharan australia china europe india africa africataiwan us A*02:01 97.8% 39.5% 94.0% 53.9% 73.3% 56.3% 35.1% 79.4%A*02:02 0.0% 0.1% 0.3% 0.9% 9.7% 24.1% 0.0% 3.6% A*02:03 0.0% 15.3% 0.2%4.9% 0.0% 0.4% 19.3% 2.2% A*02:04 0.0% 0.1% 0.0% 0.3% 2.6% 0.4% 0.0%0.2% A*02:05 1.1% 0.9% 3.2% 5.8% 13.8% 15.9% 0.1% 4.5% A*02:06 0.0%16.0% 0.9% 10.6% 0.0% 0.7% 12.8% 5.5% A*02:07 1.1% 26.1% 0.4% 0.4% 0.0%0.0% 32.7% 2.4% A*02:08 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% A*02:090.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% A*02:10 0.0% 1.1% 0.0% 0.0% 0.0%0.2% 0.0% 0.1% A*02:11 0.0% 0.1% 0.1% 22.3% 0.0% 1.5% 0.0% 1.7% otherA02 subtypes 0.0% 0.7% 0.8% 0.9% 0.5% 0.5% 0.0% 0.6% (A*02:12-A*02:93)100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

TABLE 9 HLA-A02 #17-13 #52 #61 #76 #79 subtype K_(d)(nM) K_(d)(nM)K_(d)(nM) K_(d)(nM) K_(d)(nM) A*02:02 233 16.8 394 16.8 115 A*02:03 21225.7  204* 25.7* — A*02:05 183 18.6 2000  18.6 686 A*02:06 26.4 9.42200* 9.4* 63.8 A*02:07 373 6.9 3200* 6.9* 387 A*02:11 11.3 10.4 43310.4 68.5 *low fitting confidence

Example 6. Generation of AFP158/HLA-A*02:01 Specific Chimeric AntigenReceptor-Presenting T Cells (CAR-T)

Chimeric antigen receptor therapy (CAR-T therapy) is a new form oftargeted immunotherapy. It merges the exquisite targeting specificity ofmonoclonal antibodies with the potent cytotoxicity and long-termpersistence provided by cytotoxic T cells. This technology enables Tcells to acquire long-term novel antigenic specificity independent ofthe endogenous TCR. Clinical trials have shown clinically significantantitumor activity of CAR-T therapy in neuroblastoma (Louis, C. U. etal., Blood 118(23):6050-6, 2011), B-ALL (Maude, S. L. et al., N Engl JMed. 371(16):1507-1517, 2014), CLL (Brentjens, R. J. et al., Blood.118(18):4817-4828, 2011), and B cell lymphoma (Kochenderfer, J. N. etal., Blood. 116(20):4099-4102, 2010). In one study, a 90% completeremission rate in 30 patients with B-ALL treated with CD19-CAR T therapywas reported (Maude et al., supra).

To further explore the potency of the AFP158/HLA-A*02:01 specificantibodies, we constructed anti-AFP158/HLA-A*02:01 scFv expressing CARsand transduced T cells with these CARs. AFP158/HLA-A*02:01 specific CARswere constructed using a lentiviral CAR expression vector.Anti-AFP158/HLA-A*02:01 scFvs were grafted onto a second generation CAR(Mackall, C. L. et al., Nat. Rev. Clin. Oncol. 11(12):693-703, 2014)with CD28 signaling domain and TCRζ engineered in cis to provideintracellular T cell stimulation signals and to activate T cells. FIG.11 provides a schematic illustration of the anti-AFP158/HLA-A*02:01 CARconstructs.

Example 7. Characterization of AFP158 CAR-T Cells AFP158 CAR Expressedin CD4+ and CD8+ Primary T Cells

Peripheral blood lymphocytes were isolated from healthy donors andtransduced with an AFP158 CAR construct encoding ananti-AFP158/HLA-A*02:01 binding moiety. Five days after transduction,AFP158 CAR-T cells and mock-transduced cells were co-stained with AFP158tetramer, and one of CD3, CD4 or CD8 antibodies and analyzed by flowcytometry. FIG. 24 shows flow cytometry results for AFP158 CAR- andmock-transfected cells indicating that the AFP158 CAR can be expressedin both CD4⁺ and CD8⁺ primary T cells.

Even Distribution of AFP158 CAR on T Cell Surface

It has been demonstrated that CAR-T cells with even cell surfacedistribution of CARs kill tumor cells more efficiently in vitro and invivo than CAR-T cells with uneven cell surface distribution of CARs, andthey exhaust much less easily after killing. The uneven distribution,such as aggregation, of CARs can lead to antigen-independent CARactivation, premature T cell exhaustion, and no anti-tumor activity invivo. Therefore, even distribution of CARs on T cell surface isdesirable. To determine the distribution of anti-AFP158/HLA-A*02:01 scFvexpressing CARs on T cell membrane, we used a conjugate AFP158peptide-HLA-A2 tetramer-PE to stain the AFP158 CAR-transduced T cells asdescribed above. Primary human CD3+ T cells were first activated byanti-CD3 and CD28 beads, and then transduced with AFP158 CAR lentivirus.On day 8 after viral transduction, AFP158 CAR-transduced T cells werestained with AFP158 peptide-HLA-A2 tetramer-PE at 2 ng/ml in thepresence of protein transport inhibitor for 30 minutes at roomtemperature. Images were acquired using an Olympus fluorescencemicroscope. We found that the distribution of AFP158 CAR on T cellmembrane is even and smooth. No punctate CAR distribution was observed.A representative image is shown in FIG. 26.

In Vitro Cytotoxicity Study of AFP158 CAR-T Cells

Lentiviruses containing AFP158/HLA-A*02:01-specific chimeric antigenreceptors were produced by transfection of 293T cells with CAR vectors.Human T-cells were used for transduction after 1-day stimulation withCD3/CD28 beads (Dynabeads®, Invitrogen) in the presence of interleukin-2at 100 U/ml. Concentrated lentiviruses were applied to T-cells inRetronectin (Takara) coated 6-well plates for 72 hours. Functionalassessment of the transduced T cells (AFP158 CAR-T cells) was performedusing LDH Cytotoxicity Assay. Effector-to-target ratios used were 5:1.

A panel of AFP158 CAR clones transduced into T cells was tested againstthe target cell lines HEPG2, SK-HEP1, and SK-HEP1-MiniG. T cellstransduced with the AFP158 CAR clones specifically killedAFP158/HLA*A02:01 positive cell lines HEPG2 and SK-HEP1-MiniG (FIG. 12).The AFP158 CAR expressing T cells killed the target-positive cancercells in a specific and highly efficient way for most of the antibodyclones tested. Target-negative, wild-type SK-HEP1 cells, however, werepoorly recognized by the same T cells, except for those transduced withclone #44, which showed some non-specific killing of wild-type SK-HEP1cells.

A large panel of cancer cell lines positive or negative for AFP158 andHLA*A02:01 was tested for killing by AFP158 CAR-T cells. Primary T cellsthat were mock-transduced or transduced with AFP158 CAR (a CAR constructencoding an exemplar anti-AFP158/HLA*A02:01 antibody) were tested fortheir ability to specifically kill AFP158/HLA*A02:01 positive cancercells, at an effector-to-target ratio at 5:1. As shown in FIG. 13,AFP158 CAR-T cells specifically killed AFP158/HLA*A02:01 positive celllines HepG2 and SK-Hep1-MiniG, but none of the other cell lines, whichare either AFP negative or HLA*A02:01 negative. Importantly, this datademonstrates that our antibody is highly specific to AFP-expressingcells and does not mediate non-specific killing of a large panel ofHLA-A02 cell lines across multiple tissues types that do not expressAFP.

AFP158 CAR-Transduced T Cells Degranulate Upon Antigen Stimulation

To further characterize the biological activities in AFP158CAR-transduced T cells, we used a flow cytometry assay to detect CD107asurface expression as a measurement of degranulation activity. AFP158CAR-transduced T cells were co-incubated with HepG2, SK-HEP-1 andSK-Hep1-MiniG cells for 4 hours in the presence of a 1:200 dilution ofanti-CD107a antibody and protein transport inhibitor cocktail(eBioscience). After co-incubation with target cells, transduced T cellswere stained with AFP158/HLA tetramers and anti-CD8. Degranulation intetramer-positive, CD8-positive T cells is shown in FIG. 25. The highestlevel of degranulation, as measured by CD107a expression, was observedupon co-incubation with SK-Hep1-MiniG, followed by HepG2, while nodegranulation was observed with the parental antigen-negative SK-HEP-1.This is consistent with the T-cell mediated cell lysis data above.

Cytokine Release

The cytokine release profile upon treatment with activated AFP158 CAR-Tcells was also examined. T cells were either mock-transduced ortransduced with an exemplar AFP158 CAR and co-incubated with targetcells as indicated. Release of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF,IFN-γ and TNF-α into the media was measured after 16 hours using theMagpix multiplex system (Luminex) with the Bio-plex Pro Human Cytokine8-plex Assay (BioRad). Cytokine concentrations were determined from astandard curve, after subtracting values from media, target cells aloneand AFP158 CAR transduced T cells alone. As shown in FIGS. 14A and 14B,cytokine release was detected only when AFP158 CAR-T cells wereco-incubated with AFP158/HLA*A02:01 positive cells: SK-Hep1-MiniG,MCF7-MiniG and HepG2, but not with AFP158/HLA*A02:01 negative cells:SK-Hep1 and MCF7. AFP158 CAR-T cells released much higher level ofcytokines when they were exposed to SK-Hep1-MiniG and MCF7-MiniG. Thisis consistent with AFP158 minigene-transduced cancer cells expressingmuch higher levels of AFP158/HLA*A02:01 on the cell surface than HepG2.Mock-transduced T cells released only trace amounts, if any, ofcytokines in the presence of AFP158/HLA*A02:01 positive cancer cells.IL-6 release after co-incubation with mock-transduced (AFP negative)SK-Hep1 is due to endogenous IL-6 expression in those cells (based ondata not shown).

In Vivo Efficacy Study of AFP158 CAR T Cells in Human HCC XenograftModel

The in vivo anti-tumor activity of AFP158 CAR-T cells was tested inseveral established human HCC xenograft models in SCID-beigeimmunocompromised mice. HepG2 was implanted subcutaneously (s.c.) overthe right flank at 2.5×10⁶ cells per mouse. When tumor volumes reachedan average of 100 mm³, mice were randomized based on tumor volume intofour groups: 1) no treatment, 2) intravenous (i.v.) treatment withmock-transduced, donor-matched T cells (mock), 3) intravenous (i.v.)treatment with an exemplar AFP158 CAR transduced T cells, and 4)intratumoral (i.t.) injection of AFP158 CAR transduced T cells. Alltreatments were administered at a dose of 10⁷ cells per mouse andrepeated every 2 weeks for a total of 3 doses.

Both control and AFP158 CAR-T cells were well-tolerated at the givendose and schedule; no treatment-related adverse responses were observedduring the study. Tumors in untreated mice, as well as control Tcell-treated mice, grew continuously until they reached a size thatrequired euthanasia. As shown in FIG. 15A, intravenous administration ofAFP158 CAR-T cells in HepG2 tumor-bearing mice resulted in delayed tumorgrowth starting 28 days after the first dose. Approximately 25% tumorgrowth inhibition was observed after day 35 post the first dose (FIG.15A). In contrast to the delayed effects of i.v. administration, i.t.injections of AFP158 CAR-T cells caused rapid, profound, and lastingtumor regression in all mice, with 80% ( 6/8) showing completeregression (FIG. 15B).

Peritoneal dissemination of HCC occurs in a subset of patients who havevery limited treatment options as a result. Therefore the anti-tumoractivity of AFP158 CAR-T cells was further tested in an establishedintraperitoneal HCC xenograft model. In this study, luciferase-taggedHepG2 cells (HepG2-luc2) were implanted intraperitoneally (i.p.) at2.5×10⁶ cells per mouse. Tumor burden was assessed weekly by measuringtumor-derived bioluminescence. One week post tumor implantation, animalswere randomized based on total bioluminescent flux into four groups: 1)no treatment, 2) treatment with i.p. injection of 10⁷ control T cells,treatment with i.p. injection of 10⁶ AFP158 CAR-T cells, and 4)treatment with i.p. injection of 10⁷ AFP158 CAR-T cells (n=6 mice pergroup). Two doses of T cells were administered to each group, two weeksapart.

As observed with i.v. and i.t. routes of administration, no clinicalsigns of adverse reactions were observed as a result of i.p. injectionsof either control or AFP158 CAR-T cells. As shown in FIGS. 16A (changeof the photon emission from tumor-bearing mice at day 70 aftertreatment) and 16B (photon emission images of the HepG2-Luc2tumor-bearing mice at day 70), the tumor burden in control Tcell-treated animals showed no difference from that observed in theuntreated control group. In contrast, mice treated with AFP158 CAR-Tcells at 10⁶ or 10⁷ cells per mouse showed robust tumor regression. Nodose-dependent anti-tumor activity was observed, indicating that bothdoses exceed the maximum efficacious dose in this model. Thus i.p.treatment with AFP158 CART cells in a model of peritoneal HCC is safe,potent and eradicated tumors effectively.

The in vivo activity of AFP158 CAR-T cells was also evaluated inSK-Hep1-MiniG s.c. xenograft model. SK-Hep1-MiniG was implantedsubcutaneously (s.c.) over the right flank at 2.5×10⁶ cells per mouse.When tumor volume reached an average of 100 mm³, mice were randomizedbased on tumor volume into three groups: 1) intravenous (i.v.) treatmentwith mock-transduced, donor-matched T cells (mock), 2) intravenous(i.v.) treatment with exemplar AFP158 CAR transduced T cells, and 3)intratumoral (i.t.) injection of the same AFP158 CAR transduced T cells.All treatments were administered at a dose of 10⁷ cells per mouse andrepeated once after 2 weeks of the first treatment. In this tumor model,i.v. administration of AFP158 CAR-T cells resulted in immediate tumorgrowth inhibition, slowing tumor growth by approximately 28% by day 31following the first dose (FIG. 17). This suggests that the delayed tumorgrowth inhibition activity of i.v. AFP158 CAR-T cell administration inHepG2 tumors is a model-specific phenomenon. Similar to the resultsobtained in HepG2 tumors, i.t. injection of AFP158 CAR-T cells in theSK-Hep1-MiniG mouse model resulted in robust and prolonged tumorregression shortly after the first dose (FIG. 17).

Example 8. Generation and Characterization of the Full-Length IgG1AFP158 Antibodies

Full-length human IgG1 of the selected phage clones were produced inHEK293 and Chinese hamster ovary (CHO) cell lines, as described(Tomimatsu, K. et al., Biosci. Biotechnol. Biochem. 73(7):1465-1469,2009) (data not shown). In brief, antibody variable regions weresubcloned into mammalian expression vectors, with matching human lambdaor kappa light chain constant region and human IgG1 constant regionsequences. Applying the same cloning strategy, we also generatedchimeric AFP158 full-length antibodies with mouse IgG1 heavy chain andlight chain constant regions. Molecular weight of the purifiedfull-length IgG antibodies was measured under both reducing andnon-reducing conditions by electrophoresis. SDS-PAGE of purified AFP158mouse chimeric IgG1 antibodies was performed to determine proteinpurity. In brief, 2 μg of the protein was mixed with 2.54 of the NuPAGELDS Sample Buffer (Life Technologies, NP0008) and brought up to 104 withdeionized water. The sample was heated at 70° C. for 10 minutes, andthen loaded onto the gel. Gel electrophoresis was performed at 180V for1 hour. Examples of SDS-PAGE are shown in FIG. 18.

AFP158 chimeric IgG1 antibody was tested for the binding towards AFP158presenting SK-HEP1 cells by flow cytometry. SK-HEP1 is an HLA-A*02:01positive and AFP negative cell line. An AFP158 minigene cassette wastransfected into SK-HEP1 cells to generate the AFP158-presentingSK-HEP1-miniG cells. 10 μg/mL of antibody was added to cells on ice for1 hour. After washing, R-PE conjugated anti-mouse IgG(H+L) (Vector Labs#EI-2007) was added to detect antibody binding. The AFP158 antibodieswere found to bind to the minigene-transfected SK-HEP1-miniG cells,while secondary control antibody alone did not bind to the same cells(FIG. 19). Binding affinity of the mouse chimeric IgG1 AFP158 antibodieswas determined by ForteBio. Converting the antibodies from monovalentBsAbs into bivalent IgG antibodies dramatically increased the bindingaffinity of AFP158 antibodies towards target antigen. The K_(d)'s of thefull-length antibodies were determined to be in the picomolar range, a100- to 1000-fold increase compared with the BsAb format. Table 10 showsexamples of the K_(d) data.

TABLE 10 Clone # k_(a) [1/Ms] k_(d) [1/s] K_(d) [nM] 1402-52 1.17E+062.35E−04 0.201 1402-61 5.13E+05 1.31E−05 0.0255 1402-76 6.32E+051.10E−04 0.173 1402-79 4.60E+05 5.33E−05 0.116

AFP158-specific and negative control (ET901) mouse chimeric IgG1 weretested for the binding towards AFP158/HLA-A*02:01, AFP recombinantprotein and free AFP158 peptide in an ELISA assay. Antibodies weretested at 3× serial dilution, starting from 100 ng/mL, for a total of 8concentrations. Biotinlyated AFP158/A*02:01 MHC was coated ontostreptavidin plates at 2 μg/mL, AFP protein was coated at 2 μg/mL andAFP158 peptide was coated at 40 ng/mL. It was confirmed that full-lengthAFP158 antibodies recognize the AFP158 peptide only in the context ofHLA-A02, and do not bind recombinant AFP protein or free AFP158 peptide(FIG. 20).

Example 9. Efficacy of AFP158-CART in Distant SK-Hep1-MiniG s.c.Xenograft Model

Example 7 showed that intratumoral administration of AFP158 CART cellsinto the subcutaneous (s.c.) liver tumor model significantly inhibitedthe growth of treated tumors in multiple xenograft models. The goal ofthe study described in this Example was to assess whether intratumoraltreatment would also affect the growth of distant s.c. tumors.

In one representative study, the following materials and procedures wereused:

1. Target tumor cell line SK-Hep1-MiniG (a.k.a. SK-Hep1-MG): Human HCCcell line SK-Hep1 expressing HLA-A*02:01 and AFP158 peptide minigene.

2. Animal: SCID-beige carries no T- and B-cells. Have functional NK,monocyte/macrophage, granulocytes and dendritic cells.

3. Animal study was carried out at a research contract lab.

4. CART Cells: human T cells were activated on Day 0; b) activated Tcells were transduced by lentivirus on Day 1; c) lentivirus and beadswere removed on Day 5; d) CAR T cells were cultured and expanded withIL-2 at 100 unit/ml; e) APC cells were isolated by depleting CD3⁺ T cellfrom PBMC. The cells are mainly monocytes and B-cells.

5. Animal study. 6-8 weeks old female SCID Beige mice were used in thisstudy. The SK-Hep1-MiniG cell line were cultured in DMEM Medium+10% FBSand 1% L-Glutamine at 37° C. in a humidified atmosphere with 5% CO₂.SK-Hep1-MiniG cells were resuspended in 50% PBS plus 50% Matrigel andimplanted subcutaneously at both right and left flanks into 40 mice at5×10⁶ cells/100 ul/injection site.

When tumors reached 100 mm³ on average, mice were randomized based ontumor size at right flank into the 6 groups described below, at 6 miceper group, and samples were injected into the tumors on the right flankof each mouse. For the AFP158-CART groups (groups 4-6), various antibodyclones as described above were used in various experiments. Results froman experiment using a representative clone are shown below. Other clonesproduced similar results (data not shown).

Group 1: Vehicle (PBS), 100 μL/mouse, i.t. into the right site s.c.tumor, single dose.

Group 2: Mock* T-Cells 7 Million/100 μL/mouse, i.t. into the right sites.c. tumor, single dose. (Mock T-cells are the T-cells without CARTtransduction)

Group 3: Mock with APC Cells 7 Million (70% Mock+30% APC)/100 μL/mousei. t. into the right site s.c. tumor, single dose.

Group 4: AFP158 CART Cells 7 Million/100 μL/mouse, i.v. via tail vein,single dose.

Group 5: AFP158 CART Cells 7 Million/100 μL/mouse, i.t. into the rightsite s.c. tumor, single dose.

Group 6: AFP158 CART Cells with APC, 7 Million (70% CART+30% APC)/100μL/mouse, i. t. into the right site s.c. tumor, single dose.

AFP158-CART is Safe at the Current Dose/Schedule

After dosing, body weight and other clinical behavior were closelymonitored. As shown in FIG. 21, body weight loss was not observed in anyof the groups. No other clinical signs of drug related toxicity wereobserved.

Single i. t. Dose Resulted in Regression of Local Tumors and Inhibitionof Distant Tumors

As shown in FIG. 22, a single i.t. dose of AFP158-CART injected to onlythe right flank resulted in significant inhibition of tumors implantedon both the right flank and left flank (the side which AFP158-CAR Tcells were not directly injected). Analysis by area under curve,reflecting the overall growth kinetics of the tumor, showed that i.t.injection of AFP158-CART alone resulted in 42% tumor growth inhibitionon the right flank (Dunnett's test p<0.01) and 33% tumor growthinhibition on the left flank (p>0.05).

AFP158-CART+APC Enhanced Anti-Tumor Activity in Both Sides

As shown in FIG. 22, i.t. injection of AFP158-CART plus APC causedregression of right flank tumors with overall tumor inhibition of 68%when compared with the vehicle-treated group (p<0.001). A strongerinhibition of distant left flank tumor as compared to the respectivevehicle-treated group was also observed (47%, p<0.01). This suggests thepossible involvement of T-cell cross-priming in the additionalinhibition of both right and left side tumors.

CD3 Positive Cells were Observed in Both Right and Left Side Tumors inAFP158-CART+APC Treated Animals

At the end of the study (34 days post dosing), tumors were harvested andhistological staining of CD3 (T-cell marker) was performed. As shown inFIG. 23, CD3 positive cells were found in both right and left sidetumors from i.t. injection of AFP158-CART plus APC cells. This suggeststhe involvement of the human T-cells in the tumor growth inhibition.

Sequence Listing hAFP protein (SEQ ID NO: 1)MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLI SKTRAALGV hAFP CDS(SEQ ID NO: 2) ATGAAGTGGGTGGAATCAATTTTTTTAATTTTCCTACTAAATTTTACTGAATCCAGAACACTGCATAGAAATGAATATGGAATAGCTTCCATATTGGATTCTTACCAATGTACTGCAGAGATAAGTTTAGCTGACCTGGCTACCATATTTTTTGCCCAGTTTGTTCAAGAAGCCACTTACAAGGAAGTAAGCAAAATGGTGAAAGATGCATTGACTGCAATTGAGAAACCCACTGGAGATGAACAGTCTTCAGGGTGTTTAGAAAACCAGCTACCTGCCTTTCTGGAAGAACTTTGCCATGAGAAAGAAATTTTGGAGAAGTACGGACATTCAGACTGCTGCAGCCAAAGTGAAGAGGGAAGACATAACTGTTTTCTTGCACACAAAAAGCCCACTCCAGCATCGATCCCACTTTTCCAAGTTCCAGAACCTGTCACAAGCTGTGAAGCATATGAAGAAGACAGGGAGACATTCATGAACAAATTCATTTATGAGATAGCAAGAAGGCATCCCTTCCTGTATGCACCTACAATTCTTCTTTGGGCTGCTCGCTATGACAAAATAATTCCATCTTGCTGCAAAGCTGAAAATGCAGTTGAATGCTTCCAAACAAAGGCAGCAACAGTTACAAAAGAATTAAGAGAAAGCAGCTTGTTAAATCAACATGCATGTGCAGTAATGAAAAATTTTGGGACCCGAACTTTCCAAGCCATAACTGTTACTAAACTGAGTCAGAAGTTTACCAAAGTTAATTTTACTGAAATCCAGAAACTAGTCCTGGATGTGGCCCATGTACATGAGCACTGTTGCAGAGGAGATGTGCTGGATTGTCTGCAGGATGGGGAAAAAATCATGTCCTACATATGTTCTCAACAAGACACTCTGTCAAACAAAATAACAGAATGCTGCAAACTGACCACGCTGGAACGTGGTCAATGTATAATTCATGCAGAAAATGATGAAAAACCTGAAGGTCTATCTCCAAATCTAAACAGGTTTTTAGGAGATAGAGATTTTAACCAATTTTCTTCAGGGGAAAAAAATATCTTCTTGGCAAGTTTTGTTCATGAATATTCAAGAAGACATCCTCAGCTTGCTGTCTCAGTAATTCTAAGAGTTGCTAAAGGATACCAGGAGTTATTGGAGAAGTGTTTCCAGACTGAAAACCCTCTTGAATGCCAAGATAAAGGAGAAGAAGAATTACAGAAATACATCCAGGAGAGCCAAGCATTGGCAAAGCGAAGCTGCGGCCTCTTCCAGAAACTAGGAGAATATTACTTACAAAATGCGTTTCTCGTTGCTTACACAAAGAAAGCCCCCCAGCTGACCTCGTCGGAGCTGATGGCCATCACCAGAAAAATGGCAGCCACAGCAGCCACTTGTTGCCAACTCAGTGAGGACAAACTATTGGCCTGTGGCGAGGGAGCGGCTGACATTATTATCGGACACTTATGTATCAGACATGAAATGACTCCAGTAAACCCTGGTGTTGGCCAGTGCTGCACTTCTTCATATGCCAACAGGAGGCCATGCTTCAGCAGCTTGGTGGTGGATGAAACATATGTCCCTCCTGCATTCTCTGATGACAAGTTCATTTTCCATAAGGATCTGTGCCAAGCTCAGGGTGTAGCGCTGCAAACAATGAAGCAAGAGTTTCTCATTAACCTTGTGAAGCAAAAGCCACAAATAACAGAGGAACAACTTGAGGCTGTCATTGCAGATTTCTCAGGCCTGTTGGAGAAATGCTGCCAAGGCCAGGAACAGGAAGTCTGCTTTGCTGAAGAGGGACAAAAACTGATTTCAAAAACTCGTGCTGCTTTGGGAGTTTAA hAFP137-145 (SEQ ID NO: 3) PLFQVPEPVhAFP158-166 (SEQ ID NO: 4) FMNKFIYEI hAFP325-334 (SEQ ID NO: 5)GLSPNLNRFL hAFP542-550 (SEQ ID NO: 6) GVALQTMKQ hAFP158 A1(SEQ ID NO: 7) AMNKFIYEI hAFP158 A3 (SEQ ID NO: 8) FMAKFIYEI hAFP158 A4(SEQ ID NO: 9) FMNAFIYEI hAFP158 A5 (SEQ ID NO: 10) FMNKAIYEI hAFP158 A6(SEQ ID NO: 11) FMNKFAYEI hAFP158 A7 (SEQ ID NO: 12) FMNKFIAEIhAFP158 A8 (SEQ ID NO: 13) FMNKFIYAI mAFP protein (SEQ ID NO: 14)MKWITPASLILLLHFAASKALHENEFGIASTLDSSQCVTEKNVLSIATITFTQFVPEATEEEVNKMTSDVLAAMKKNSGDGCLESQLSVFLDEICHETELSNKYGLSGCCSQSGVERHQCLLARKKTAPASVPPFQFPEPAESCKAHEENRAVFMNRFIYEVSRRNPFMYAPAILSLAAQYDKVVLACCKADNKEECFQTKRASIAKELREGSMLNEHVCSVIRKFGSRNLQATTIIKLSQKLTEANFTEIQKLALDVAHIHEECCQGNSLECLQDGEKVMTYICSQQNILSSKIAECCKLPMIQLGFCIIHAENGVKPEGLSLNPSQFLGDRNFAQFSSEEKIMFMASFLHEYSRTHPNLPVSVILRIAKTYQEILEKCSQSGNLPGCQDNLEEELQKHIEESQALSKQSCALYQTLGDYKLQNLFLIGYTRKAPQLTSAELIDLTGKMVSIASTCCQLSEEKWSGCGEGMADIFIGHLCIRNEASPVNSGISHCCNSSYSNRRLCITSFLRDETYAPPPFSEDKFIFHKDLCQAQGKALQTMKQELLINLVKQKPELTEEQLAAVTADFSGLLEKCCKAQDQEVCFTEEGPKLISKTR DALGV mAFP CDS(SEQ ID NO: 15) ATGAAGTGGATCACACCCGCTTCCCTCATCCTCCTGCTACATTTCGCTGCGTCCAAAGCATTGCACGAAAATGAGTTTGGGATAGCTTCCACGTTAGATTCCTCCCAGTGCGTGACGGAGAAGAATGTGCTTAGCATAGCTACCATCACCTTTACCCAGTTTGTTCCGGAAGCCACCGAGGAGGAAGTGAACAAAATGACTAGCGATGTGTTGGCTGCAATGAAGAAAAACTCTGGCGATGGGTGTTTAGAAAGCCAGCTATCTGTGTTTCTGGATGAAATTTGTCATGAGACGGAACTCTCTAACAAGTATGGACTCTCAGGCTGCTGCAGCCAAAGTGGAGTGGAAAGACATCAGTGTCTGCTGGCACGCAAGAAGACTGCTCCGGCCTCTGTCCCACCCTTCCAGTTTCCAGAACCTGCCGAGAGTTGCAAAGCACATGAAGAAAACAGGGCAGTGTTCATGAACAGGTTCATCTATGAAGTGTCAAGGAGGAACCCCTTCATGTATGCCCCAGCCATTCTGTCCTTGGCTGCTCAGTACGACAAGGTCGTTCTGGCATGCTGCAAAGCTGACAACAAGGAGGAGTGCTTCCAGACAAAGAGAGCATCCATTGCAAAGGAATTAAGAGAAGGAAGCATGTTAAATGAGCATGTATGTTCAGTGATAAGAAAATTTGGATCCCGAAACCTCCAGGCAACAACCATTATTAAGCTAAGTCAAAAGTTAACTGAAGCAAATTTTACTGAGATTCAGAAGCTGGCCCTGGATGTGGCTCACATCCACGAGGAGTGTTGCCAAGGAAACTCGCTGGAGTGTCTGCAGGATGGGGAAAAAGTCATGACATATATATGTTCTCAACAAAATATTCTGTCAAGCAAAATAGCAGAGTGCTGCAAATTACCCATGATCCAACTAGGCTTCTGCATAATTCACGCAGAGAATGGCGTCAAACCTGAAGGCTTATCTCTAAATCCAAGCCAGTTTTTGGGAGACAGAAATTTTGCCCAATTTTCTTCAGAGGAAAAAATCATGTTCATGGCAAGCTTTCTTCATGAATACTCAAGAACTCACCCCAACCTTCCTGTCTCAGTCATTCTAAGAATTGCTAAAACGTACCAGGAAATATTGGAGAAGTGTTCCCAGTCTGGAAATCTACCTGGATGTCAGGACAATCTGGAAGAAGAATTGCAGAAACACATCGAGGAGAGCCAGGCACTGTCCAAGCAAAGCTGCGCTCTCTACCAGACCTTAGGAGACTACAAATTACAAAATCTGTTCCTTATTGGTTACACGAGGAAAGCCCCTCAGCTGACCTCAGCAGAGCTGATCGACCTCACCGGGAAGATGGTGAGCATTGCCTCCACGTGCTGCCAGCTCAGCGAGGAGAAATGGTCCGGCTGTGGTGAGGGAATGGCCGACATTTTCATTGGACATTTGTGTATAAGGAATGAAGCAAGCCCTGTGAACTCTGGTATCAGCCACTGCTGCAACTCTTCGTATTCCAACAGGAGGCTATGCATCACCAGTTTTCTGAGGGATGAAACCTATGCCCCTCCCCCATTCTCTGAGGATAAATTCATCTTCCACAAGGATCTGTGCCAAGCTCAGGGCAAAGCCCTACAGACCATGAAACAAGAGCTTCTCATTAACCTGGTGAAGCAAAAGCCTGAACTGACAGAGGAGCAGCTGGCGGCTGTCACTGCAGATTTCTCGGGCCTTTTGGAGAAGTGCTGCAAAGCCCAGGACCAGGAAGTCTGTTTCACAGAAGAGGGTCCAAAGTTGATTTCCAAAACTCGT GATGCTTTGGGCGTTTAAmAFP154-162 (SEQ ID NO: 16) FMNRFIYEVClone 17 heavy chain variable domain, protein (SEQ ID NO: 17)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWSAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARYQD WWYLGQFDQWGQGTLVTVSSClone 33 heavy chain variable domain, protein (SEQ ID NO: 18)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGSYYSGRYDAWGQGTLVTVSS Clone 44 heavy chain variable domain, protein(SEQ ID NO: 19) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREIRGYYYYYGMDVWGQGTTVTVSS Clone 48 heavy chain variable domain, protein(SEQ ID NO: 20) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARADDYGAPYYYYGMDVWGQGTTVTVSS Clone 50 heavy chain variable domain, protein(SEQ ID NO: 21) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCATGY GGYFDYWGQGTLVTVSSClone 52 heavy chain variable domain, protein (SEQ ID NO: 22)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDS YYYYYGMDVWGQGTTVTVSSClone 61 heavy chain variable domain, protein (SEQ ID NO: 23)EVQLVQSGAEVKKPGESLTISCKASGYSFPNYWITWVRQMSGGGLEWMGRIDPGDSYTTYNPSFQGHVTISIDKSTNTAYLHWNSLKASDTAMYYCARYY VSLVDIWGQGTLVTVSSClone 76 heavy chain variable domain, protein (SEQ ID NO: 24)EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNNLKTEDTAVYYCARDGLYSSSWYDSDYWGQGTLVTVSS Clone 79 heavy chain variable domain, protein(SEQ ID NO: 25) QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIHSGSYYGLLYYAMDVWGQGTTVTVSSClone 17-13 heavy chain variable domain, protein (SEQ ID NO: 26)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARFQ DWWYLGQFDQWGQGTLVTVSSClone 17 1ight chain variable domain, protein (SEQ ID NO: 27)QSALTQPASVSGSPGQSITISCTATGSDVGVYYYVSWYQQHPGKAPKVMIYDVGNRPPGVSNRFSGSKSGNTASLTISGLQAEDEADYYCASYTNRNSLG YVFGTGTKVTVLGClone 33 1ight chain variable domain, protein (SEQ ID NO: 28)NFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSTVV FGGGTKLTVLGClone 44 1ight chain variable domain, protein (SEQ ID NO: 29)SYELTQPPSVSVAPGKTARITCGGDNIGTKSVTWYQQRPGQAPMMVIYYDTVRPSGIPERLSGSNSGNTATLTITRVEAGDEADYYCQVWDSSSDHPVFG GGTKLTVLGClone 48 1ight chain variable domain, protein (SEQ ID NO: 30)QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGS VFGTGTKVTVLGClone 50 light chain variable domain, protein (SEQ ID NO: 31)QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFG GGTKLTVLGClone 52 1ight chain variable domain, protein (SEQ ID NO: 32)QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPYWFQQKPGQAPRTLIYDASDKHSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCLLSYSDALVF GGGTKLTVLGClone 61 1ight chain variable domain, protein (SEQ ID NO: 33)QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSEVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTGSRAV FGGGTKLTVLGClone 76 1ight chain variable domain, protein (SEQ ID NO: 34)QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDGSLYTML FGGGTKLTVLGClone 79 1ight chain variable domain, protein (SEQ ID NO: 35)QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIFGNSNRPSGVPDRFSGFKSGTSASLAITGLQAEDEADYFCQSYDSSLSGS GVFGTGTKVTVLGClone 17-13 1ight chain variable domain, protein (SEQ ID NO: 36)QSALTQPASVSGSPGQSITISCTATGSDVGVYYYVSWYQQHPGKAPKVMIYDVDNRPPGVSNRFSGSKSGNTASLTISGLQAEDEADYYCASYTNRNSLG YVFGTGTKVTVLGClone 17 heavy chain variable domain, nucleic acid (SEQ ID NO: 37)GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGCGCTACCAGGACTGGTGGTACCTGGGTCAGTTCGATCAGTGGGGTCAAGGTACTCTGGT GACCGTCTCCTCAClone 33 heavy chain variable domain, nucleic acid (SEQ ID NO: 38)CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATTTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCGCGCGGTTCTTACTACTCTGGTCGTTACGATGCTTGGGGTCAAGGTACTCT GGTGACCGTCTCCTCAClone 44 heavy chain variable domain, nucleic acid (SEQ ID NO: 39)CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAAATTAGGGGCTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCAClone 48 heavy chain variable domain, nucleic acid (SEQ ID NO: 40)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAACAGTGGTAGAATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGATGACTACGGCGCCCCCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAClone 50 heavy chain variable domain, nucleic acid (SEQ ID NO: 41)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGACCGGTTATGGGGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGNCACCGTCTCCTC AClone 52 heavy chain variable domain, nucleic acid (SEQ ID NO: 42)GAAGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATTCCTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCCTCAClone 61 heavy chain variable domain, nucleic acid (SEQ ID NO: 43)GAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAGCCTGGCGAGAGCCTGACCATCTCCTGCAAGGCCAGCGGCTACAGCTTCCCCAACTACTGGATCACCTGGGTGCGCCAGATGTCCGGCGGAGGCCTGGAATGGATGGGCAGAATCGACCCCGGCGACAGCTACACAACCTACAACCCCAGCTTCCAGGGCCACGTGACCATCAGCATCGACAAGAGCACCAATACCGCCTACCTGCACTGGAACAGCCTGAAGGCCTCCGACACCGCCATGTACTACTGCGCCCGGTACTATGTGTCCCTGGTGGATATCTGGGGCCAGGGCACACTCGTGACCGTGTCTAG CClone 76 heavy chain variable domain, nucleic acid (SEQ ID NO: 44)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATACGCCGCCTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGCAAATGAACAACCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCTAGAGATGGGCTGTATAGCAGCAGCTGGTACGATTCTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAClone 79 heavy chain variable domain, nucleic acid (SEQ ID NO: 45)CAGATGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATCCATAGTGGGAGCTACTACGGCCTACTCTACTACGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAClone 17-13 heavy chain variable domain, nucleic acid (SEQ ID NO: 46)GAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACGGCATCAGCTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCTGGATCAGCGCCTACAACGGCAATACCAACTACGCCCAGAAACTGCAGGGCAGAGTGACCATGACCACCGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTATTGCGCCCGGTTCCAGGACTGGTGGTATCTGGGCCAGTTCGACCAGTGGGGCCAGGGCACACTCGT GACCGTGTCTAGCClone 17 1ight chain variable domain, nucleic acid (SEQ ID NO: 47)CAATCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGCAACCGGCAGTGACGTTGGTGTTTATTACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAAGTGATGATTTATGATGTCGGTAATCGGCCCCCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCGCCTCATATACAAACAGGAACAGTCTCGGCTATGTCTTCGGAACCGGGACCAAGGTCACCGTCCTAGGClone 33 1ight chain variable domain, nucleic acid (SEQ ID NO: 48)AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCACCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTClone 44 1ight chain variable domain, nucleic acid (SEQ ID NO: 49)TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCTGGCAAGACGGCCAGGATTACCTGTGGGGGTGACAACATTGGAACTAAAAGTGTGACCTGGTACCAACAGAGGCCAGGCCAGGCCCCTATGATGGTCATCTATTATGATACCGTCCGGCCCTCAGGGATCCCTGAGCGACTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCACCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATCCGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTClone 48 1ight chain variable domain, nucleic acid (SEQ ID NO: 50)CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCGGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTCAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGGTClone 50 1ight chain variable domain, nucleic acid (SEQ ID NO: 51)CAGTCTGTGTTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTClone 52 1ight chain variable domain, nucleic acid (SEQ ID NO: 52)CAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGCACTGGAGCTGTCACCAGTGGTCATTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATGCAAGCGACAAACACTCCTGGACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGGGCAAAGCTGCCCTGACCCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGAGTATTACTGCTTGCTCTCCTATAGTGATGCTCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTClone 61 1ight chain variable domain, nucleic acid (SEQ ID NO: 53)CAGAGCGTGCTGACACAGCCTGCCTCCGTGTCTGGCTCTCCTGGCCAGTCCATCACCATCAGCTGTACCGGCACCAGCTCCGACGTGGGCGGCTACAATTACGTGTCCTGGTATCAGCAGCATCCCGGCAAGGCCCCCAAGCTGATGATCTACGACGTGAACAACCGGCCCAGCGAGGTGTCCAACAGATTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACAATCAGCGGACTGCAGGCCGAGGACGAGGCCGACTACTACTGCAGCAGCTACACCACCGGCAGCAGAGCCGTGTTTGGCGGAGGCACCAAGCTGACAGTGCTGGGCClone 76 light chain variable domain, nucleic acid (SEQ ID NO: 54)CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTACTACTGCGGAACATGGGATGGCAGCCTCTATACTATGTTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTClone 79 1ight chain variable domain, nucleic acid (SEQ ID NO: 55)CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTTTGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTTCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGACTATTTCTGCCAGTCGTATGACAGTAGCCTGAGTGGTTCGGGGGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGGTClone 17-13 1ight chain variable domain, nucleic acid (SEQ ID NO: 56)CAGAGCGCCCTGACACAGCCTGCCTCCGTGTCTGGATCTCCCGGCCAGAGCATCACCATCAGCTGCACAGCCACCGGCTCCGACGTGGGCGTGTACTACTACGTGTCCTGGTATCAGCAGCATCCCGGCAAGGCCCCCAAAGTGATGATCTACGACGTGGACAACCGGCCTCCCGGCGTGTCCAATAGATTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCCTGACAATCAGCGGACTGCAGGCCGAGGACGAGGCCGATTACTACTGCGCCAGCTACACCAACCGGAACAGCCTGGGCTACGTGTTCGGCACCGGCACCAAAGTGACAGTGCTGGGC Clone 17 HCDR1 (SEQ ID NO: 57)GYTFTSYG Clone 33 HCDR1 (SEQ ID NO: 58) VSSNSAAWN Clone 44 HCDR1(SEQ ID NO: 59) GGTFSSYA Clone 48 HCDR1 (SEQ ID NO: 60) GFTFDDYAClone 50 HCDR1 (SEQ ID NO: 61) GGSISSSNW Clone 52 HCDR1 (SEQ ID NO: 62)GYTFTSYG Clone 61 HCDR1 (SEQ ID NO: 63) GYSFPNYW Clone 76 HCDR1(SEQ ID NO: 64) GFTFSNAW Clone 79 HCDR1 (SEQ ID NO: 65) GFTFDDYAClone 17-13 HCDR1 (SEQ ID NO: 66) GYTFTSYG Clone 17 HCDR2(SEQ ID NO: 67) ISAYNGNT Clone 33 HCDR2 (SEQ ID NO: 68) YRSKWYNClone 44 HCDR2 (SEQ ID NO: 69) IIPIFGTA Clone 48 HCDR2 (SEQ ID NO: 70)ISWNSGRI Clone 50 HCDR2 (SEQ ID NO: 71) IYHSGST Clone 52 HCDR2(SEQ ID NO: 72) ISAYNGNT Clone 61 HCDR2 (SEQ ID NO: 73) IDPGDSYTClone 76 HCDR2 (SEQ ID NO: 74) IRSKAYGGTT Clone 79 HCDR2 (SEQ ID NO: 75) ISWNSGSI Clone 17-13 HCDR2 (SEQ ID NO: 76) ISAYNGNTClone 17 HCDR3 (SEQ ID NO: 77) ARYQDWWYLGQFDQ Clone 33 HCDR3(SEQ ID NO: 78) ARGSYYSGRYDA Clone 44 HCDR3 (SEQ ID NO: 79)AREIRGYYYYYGMDV Clone 48 HCDR3 (SEQ ID NO: 80) ARADDYGAPYYYYGMDVClone 50 HCDR3 (SEQ ID NO: 81) ATGYGGYFDY Clone 52 HCDR3 (SEQ ID NO: 82)ARDSYYYYYGMDV Clone 61 HCDR3 (SEQ ID NO: 83) ARYYVSLVDI Clone 76 HCDR3(SEQ ID NO: 84) ARDGLYSSSWYDSDY Clone 79 HCDR3 (SEQ ID NO: 85)AKDIHSGSYYGLLYYAMDV Clone 17-13 HCDR3 (SEQ ID NO: 86) ARFQDWWYLGQFDQHCCDR1 consensus (SEQ ID NO: 87) G-F/Y-S/T-F-D/S/T-D/N/S-Y/A-A/G/WHCCDR2 consensus (SEQ ID NO: 88) I/S-K/S-X-H/Y-X-G-X-T HCCDR3 consensus(SEQ ID NO: 89) A/G-X-W/Y-Y-X-X-X-F/Y-D Clone 17 LCDR1 (SEQ ID NO: 90)GSDVGVYYY Clone 33 LCDR1 (SEQ ID NO: 91) SGSIASNY Clone 44 LCDR1(SEQ ID NO: 92) NIGTKS Clone 48 LCDR1 (SEQ ID NO: 93) SSNIGAGYDClone 50 LCDR1 (SEQ ID NO: 94) NIGSKS Clone 52 LCDR1 (SEQ ID NO: 95)TGAVTSGHY Clone 61 LCDR1 (SEQ ID NO: 96) SSDVGGYNY Clone 76 LCDR1(SEQ ID NO: 97) SSNIGNNY Clone 79 LCDR1 (SEQ ID NO: 98) SSNIGAGYDClone 17-13 LCDR1 (SEQ ID NO: 99) GSDVGVYYY Clone 17 LCDR2(SEQ ID NO: 100) DVG Clone 33 LCDR2 (SEQ ID NO: 101) EDN Clone 44 LCDR2(SEQ ID NO: 102) YDT Clone 48 LCDR2 (SEQ ID NO: 103) GNS Clone 50 LCDR2(SEQ ID NO: 104) YDS Clone 52 LCDR2 (SEQ ID NO: 105) DAS Clone 61 LCDR2(SEQ ID NO: 106) DVN Clone 76 LCDR2 (SEQ ID NO: 107) DNN Clone 79 LCDR2(SEQ ID NO: 108) GNS Clone 17-13 LCDR2 (SEQ ID NO: 109) DVDClone 17 LCDR3 (SEQ ID NO: 110) ASYTNRNSLGYV Clone 33 LCDR3(SEQ ID NO: 111) QSYDSSTVV Clone 44 LCDR3 (SEQ ID NO: 112) QVWDSSSDHPVClone 48 LCDR3 (SEQ ID NO: 113) QSYDSSLSGSV Clone 50 LCDR3(SEQ ID NO: 114) QVWDSSSDHVV Clone 52 LCDR3 (SEQ ID NO: 115) LLSYSDALVClone 61 LCDR3 (SEQ ID NO: 116) SSYTTGSRAV Clone 76 LCDR3(SEQ ID NO: 117) GTWDGSLYTML Clone 79 LCDR3 (SEQ ID NO: 118)QSYDSSLSGSGV Clone 17-13 LCDR3 (SEQ ID NO: 119) ASYTNRNSLGYVLCCDR1 consensus (SEQ ID NO: 120) S/T-G/S-D/N-I/V-A/G-A/S/V-X-H/YLCCDR3 consensus (SEQ ID NO: 121) Q-S/T-Y/W-D/T-S/T-A/S IFI30 control(SEQ ID NO: 122) LLDVPTAAV BTG2 control (SEQ ID NO: 123) TLWVDPYEVBCR control (SEQ ID NO: 124) FLLDHLKRV IFI30 control (SEQ ID NO: 125)LLLDVPTAAV SSR1 control  (SEQ ID NO: 126) VLFRGGPRGLLAV PPP2R1B control(SEQ ID NO: 127) SLLPAIVEL DDX5 control (SEQ ID NO: 128) YLLPAIVHICTSG control (SEQ ID NO: 129) FLLPTGAEA CD247 control (SEQ ID NO: 130)LLDPKLCYLL DMTN control (SEQ ID NO: 131) SLPHFHHPET CALR control(SEQ ID NO: 132) MLLSVPLLLG PIM1 control (SEQ ID NO: 133) LLYDMVCGDIPIFI30 control (SEQ ID NO: 134) LLLDVPTAAVQ IFI30 control(SEQ ID NO: 135) LLLDVPTAAVQA SSR1 control (SEQ ID NO: 136)VLFRGGPRGLLAVA HLA-E control (SEQ ID NO: 137) MVDGTLLLL RPS6KB1 control(SEQ ID NO: 138) YMAPEILMRS CSF2RA control (SEQ ID NO: 139) FIYNADLMNCIL7 control (SEQ ID NO: 140) KQYESVLMVSI hTERT540 control(SEQ ID NO: 141) ILAKFLHWL

1-34. (canceled) 35: A method of directing effector cells to kill atarget cell expressing a complex comprising an alpha-fetoprotein (AFP)peptide and a major histocompatibility (MHC) class I protein (an AFP/MHCclass I complex, or AMC) in an individual, comprising administering tothe individual an effective amount of an effector cell comprising ananti-AMC construct comprising an extracellular domain comprising ananti-AMC antibody moiety that specifically binds to the AFP/MHC class Icomplex, wherein the AFP peptide has the amino acid sequence of SEQ IDNO:
 4. 36: The method of claim 35, wherein the MHC class I protein isHLA-A02. 37: The method of claim 36, wherein the MHC class I protein isthe HLA-A*02:01 subtype of the HLA-A02 allele. 38: The method of claim35, wherein the anti-AMC construct binds to the AFP/MHC class I complexwith a K_(d) from about 0.1 pM to about 500 nM. 39: The method of claim35, wherein the anti-AMC antibody moiety in monovalent scFv format bindsto the AFP/MHC class I complex with a Kd from about 10 to about 500 nM.40: The method of claim 35, wherein the anti-AMC construct is a chimericantigen receptor (CAR) comprising an extracellular domain comprising theantibody moiety, a transmembrane domain, and an intracellular signalingdomain capable of activating the effector cell. 41: The method of claim40, wherein the intracellular signaling domain comprises a CD3ζintracellular signaling sequence and a CD28 intracellular signalingsequence. 42: The method of claim 35, wherein the effector cell is a Tcell. 43: A method of treating an AFP-positive cancer in an individual,comprising subjecting the individual to the method of claim
 35. 44: Themethod of claim 43, wherein the AFP-positive cancer is hepatocellularcarcinoma, germ cell tumor, or breast cancer. 45: The method of claim35, wherein the effector cell is via intravenous route or intratumoralroute. 46: The method of claim 43, wherein the cancer is hepatocellularcarcinoma. 47: The method of claim 43, wherein the AFP-positive diseaseis hepatocellular carcinoma and metastasis is inhibited. 48: A method ofdirecting effector T cells to kill a target cell expressing an AFP/MHCclass I complex in an individual, comprising administering to theindividual an effective amount of a multi-specific anti-AMC constructcomprising: a) an anti-AMC antibody moiety that specifically binds tothe AFP/MHC class I complex, wherein the AFP peptide has the amino acidsequence of SEQ ID NO: 4, and b) a second binding moiety thatspecifically binds to an effector T cell. 49: The method of claim 48,wherein the second binding moiety specifically binds to CD3ε. 50: Themethod of claim 49, wherein the anti-AMC construct is a tandem scFvcomprising an N-terminal scFv specific for the AFP/MHC class I complexand a C-terminal scFv specific for CD3ε. 51: The method of claim 48,wherein the anti-AMC construct binds to the AFP/MHC class I complex witha K_(d) from about 0.1 pM to about 500 nM. 52: The method of claim 48,wherein the anti-AMC antibody moiety in monovalent scFv format binds tothe AFP/MHC class I complex with a Kd from about 10 to about 500 nM. 53:The method of claim 48, wherein the MEW class I protein is HLA-A02. 54:The method of claim 50, wherein the method treats an AFP-positive cancerin the individual.